Piperidine derivatives as HDAC1/2 inhibitors

ABSTRACT

Provided herein are compounds, pharmaceutical compositions comprising such compounds, and methods of using such compounds to treat diseases or disorders associated with HDAC1 and/or HDAC2 activity.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.16/433,386, filed Jun. 6, 2019, which application is a continuation ofU.S. patent application Ser. No. 15/700,998, filed Sep. 11, 2017, nowU.S. Pat. No. 10,358,421, which application is a divisional of U.S.patent application Ser. No. 14/966,556, filed Dec. 11, 2015, now U.S.Pat. No. 9,790,180, which application claims priority to U.S.Provisional Application Ser. No. 62/091,221, filed Dec. 12, 2014, and toU.S. Provisional Application Ser. No. 62/238,931, filed Oct. 8, 2015,which are incorporated herein by reference in their entireties.

BACKGROUND

A biological target of current interest is histone deacetylase (HDAC)(see, for example, a discussion of the use of inhibitors of histonedeacetylases for the treatment of cancer: Marks et al. Nature ReviewsCancer 2001, 7, 194; Johnstone et al. Nature Reviews Drug Discovery2002, 287). Post-translational modification of proteins throughacetylation and deacetylation of lysine residues plays a critical rolein regulating their cellular functions. HDACs are zinc hydrolases thatmodulate gene expression through deacetylation of the N-acetyl-lysineresidues of histone proteins and other transcriptional regulators(Hassig et al. Curr. Opin. Chem. Biol. 1997, 1, 300-308). HDACsparticipate in cellular pathways that control cell shape anddifferentiation, and an HDAC inhibitor has been shown to be effective intreating an otherwise recalcitrant cancer (Warrell et al. J. Natl.Cancer Inst. 1998, 90, 1621-1625).

Eleven human HDACs, which use Zn as a cofactor, have been identified(Taunton et al. Science 1996, 272, 408-411; Yang et al. J. Biol. Chem.1997, 272, 28001-28007. Grozinger et al. Proc. Natl. Acad. Sci. U.S.A.1999, 96, 4868-4873; Kao et al. Genes Dev. 2000, 14, 55-66. Hu et al. J.Biol. Chem. 2000, 275, 15254-15264; Zhou et al. Proc. Natl. Acad. SciU.S.A. 2001, 98, 10572-10577; Venter et al. Science 2001, 291,1304-1351) and these members fall into three classes (class I, II, andIV) based on sequence homology to their yeast orthologues (O. Witt etal. Cancer Letters, 2009, 277, 8-21). Class I HDACs include HDAC1,HDAC2, HDAC3, and HDAC8, and are referred to as “classical” HDACs, whichimplies a catalytic pocket with a Zn²⁺ ion at its base.

There remains a need for preparing structurally diverse HDAC inhibitors,particularly ones that are potent and/or selective inhibitors ofparticular classes of HDACs and individual HDACs.

SUMMARY OF THE INVENTION

Provided herein are compounds, pharmaceutical compositions comprisingsuch compounds, and methods of using such compounds to treat diseases ordisorders associated with HDAC activity, particularly diseases ordisorders that involve HDAC1 and/or HDAC2 expression. Diseases thatinvolve HDAC1 and/or HDAC2 expression include, but are not limited to,various types of cancer and hemoglobinopathies, such as sickle-cellanemia and beta-thalassemia.

Thus, in one aspect, provided herein is a compound of Formula I:

-   -   or a pharmaceutically acceptable salt thereof.

In an embodiment, provided herein is a compound of Formula II:

-   -   or a pharmaceutically acceptable salt thereof.

In a particular embodiment, provided herein is a compound of FormulaIII:

-   -   or a pharmaceutically acceptable salt thereof.

In another embodiment, provided herein are the compounds of Table 1, orpharmaceutically acceptable salts thereof.

In another aspect, provided herein is a pharmaceutical compositioncomprising a compound of Formula I, Formula II, Formula IIa, FormulaIII, or Formula IIIa, a compound presented in Table 1, a compoundpresented in Table 1a, or pharmaceutically acceptable salts thereof,together with a pharmaceutically acceptable carrier.

In another aspect, provided herein is a method of inhibiting theactivity of HDAC1 and/or HDAC2 in a subject comprising administering tothe subject a compound of Formula I, Formula II, Formula IIa, FormulaIII, or Formula IIIa, a compound presented in Table 1, a compoundpresented in Table 1a, or pharmaceutically acceptable salts thereof.

In another aspect, provided herein is a method of selectively inhibitingthe activity of each of HDAC1 and/or HDAC2 over other HDACs in a subjectcomprising administering to the subject a compound of Formula I, FormulaII, Formula IIa, Formula III, or Formula IIIa, a compound presented inTable 1, a compound presented in Table 1a, or pharmaceuticallyacceptable salts thereof. In some embodiments, the compound has aselectivity for each of HDAC1 and/or HDAC2 that is 5 to 1000 foldgreater than for other HDACs. In other embodiments, the compound has aselectivity for each of HDAC1 and/or HDAC2 when tested in a HDAC enzymeassay, of about 5 to about 1000 fold greater than for other HDACs.

In another aspect, provided herein is a method for treating a diseasemediated by one or more HDACs in a subject comprising administering tothe subject in need thereof a compound of Formula I, Formula II, FormulaIIa, Formula III, or Formula IIIa, a compound presented in Table 1, acompound presented in Table 1a, or pharmaceutically acceptable saltsthereof. In some embodiments, the disease is mediated by HDAC1 andHDAC2. In another embodiment, the disease is mediated by HDAC1. In yetanother embodiment, the disease is mediated by HDAC2.

In another aspect, provided herein is a method for treating a disease ina subject comprising administering to the subject a compound of FormulaI, Formula II, Formula IIa, Formula III, or Formula IIIa, a compoundpresented in Table 1, a compound presented in Table 1a, orpharmaceutically acceptable salts thereof. In an embodiment, the diseaseis myelodysplastic syndrome. In an embodiment, the disease is ahemoglobinopathy. In another embodiment, the disease is sickle-celldisease. In yet another embodiment, the disease is beta-thalassemia.

In a further embodiment, the disease is a cancer. The cancer can beselected from lung cancer, colon cancer, breast cancer, neuroblastoma,leukemia, or lymphoma. In yet a further embodiment, the cancer isneuroblastoma. The leukemia can be acute myelogenous leukemia or acutemegakaryocytic leukemia.

In another aspect, provided herein is a method for treating sickle celldisease, beta thalassemia, myelodysplastic syndrome, acute myelogenousleukemia, neuroblastoma, or acute megakaryocytic leukemia in a subjectin need thereof comprising administering to the subject atherapeutically effective amount of a compound of Formula I, Formula II,Formula IIa, Formula III, or Formula IIIa, a compound presented in Table1, a compound presented in Table 1a, or pharmaceutically acceptablesalts thereof.

In a further embodiment of the methods of treatment described herein,the subject to be treated is a human.

In yet another aspect, provided herein is a method for treating adisease or disorder associated with GATA binding protein 2 (Gata2)deficiency comprising administering to a subject in need thereof atherapeutically effective amount of a compound of Formula I, Formula II,Formula IIa, Formula III, or Formula IIIa, a compound presented in Table1, a compound presented in Table 1a, or pharmaceutically acceptablesalts thereof.

In still another aspect, provided herein is a method for increasing GATAbinding protein 2 (Gata2) expression in a cell comprising contacting thecell with a compound of Formula I, Formula II, Formula IIa, Formula III,or Formula IIIa, a compound presented in Table 1, a compound presentedin Table 1a, or pharmaceutically acceptable salts thereof.

In a further aspect, provided herein is a method for inducing HbG (gammaglobin) expression in a subject, comprising administering to the subjecta compound of Formula I, Formula II, Formula IIa, Formula III, orFormula IIIa, a compound presented in Table 1, a compound presented inTable 1a, or pharmaceutically acceptable salts thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the HDAC inhibition profile of Compound 003with respect to HDAC1, HDAC2, and HDAC3 (See, Example 43).

FIG. 2 shows the plasma concentration in a rat as a function of timeupon oral administration of 40 mg/kg of Compound 003 (See Example 44).

FIG. 3 shows the in vitro fetal globin induction of Compound 003 incomparison to another known HDAC1/2 inhibitor, Compound A (See Example45).

FIG. 4 is a graph showing the HDAC inhibition profile of Compound 005with respect to HDAC1, HDAC2, and HDAC3 (See, Example 43).

FIG. 5 shows the plasma concentration in a rat as a function of timeupon oral administration of 20 mg/kg of Compound 005 (See Example 44).

FIG. 6 shows the in vitro fetal globin induction of Compound 005 incomparison to another known HDAC1/2 inhibitor, Compound A (See Example45).

FIG. 7 shows that treatment of erythroid progenitors with variousHDAC1/2 inhibitors (Compounds 005 and A) leads to induction of Gata2mRNA.

DETAILED DESCRIPTION

The instant application is directed, generally, to compounds,pharmaceutical compositions comprising such compounds, and methods ofusing such compounds to treat diseases or disorders associated with HDACactivity, particularly diseases or disorders that involve any type ofHDAC1 and/or HDAC2 expression.

Definitions

Listed below are definitions of various terms used to describe thisinvention. These definitions apply to the terms as they are usedthroughout this specification and claims, unless otherwise limited inspecific instances, either individually or as part of a larger group.

The term “about” generally indicates a possible variation of no morethan 10%, 5%, or 1% of a value. For example, “about 25 mg/kg” willgenerally indicate, in its broadest sense, a value of 22.5-27.5 mg/kg,i.e., 25±2.5 mg/kg.

The term “alkyl” refers to saturated, straight- or branched-chainhydrocarbon moieties containing, in certain embodiments, between one andsix, or one and eight carbon atoms, respectively. The number of carbonatoms in an alkyl substituent can be indicated by the prefix“C_(x)-C_(y),” where x is the minimum and y is the maximum number ofcarbon atoms in the substituent. Likewise, a C_(x) chain descriptionindicates a group containing x carbon atoms (i.e., not including thenumber of heteroatoms). Examples of C₁-C₆-alkyl moieties include, butare not limited to, methyl, ethyl, propyl, isopropyl, n-butyl,tert-butyl, neopentyl, n-hexyl moieties; and examples of C₁-C₈-alkylmoieties include, but are not limited to, methyl, ethyl, propyl,isopropyl, n-butyl, tert-butyl, neopentyl, n-hexyl, heptyl, and octylmoieties.

The term “alkoxy” refers to an —O-alkyl moiety. Non-limiting examples ofC₁-C₆-alkoxy include methoxy, ethoxy, 1-propoxy, 2-propoxy, n-butoxy,t-butoxy, pentoxy, hexoxy, etc. The alkyl portion of alkoxy can bestraight- or branched-chain.

The term “aryl” refers to a mono- or poly-cyclic carbocyclic ring systemhaving one or more aromatic rings, fused or non-fused, including, butnot limited to, phenyl (i.e., C₆-aryl), naphthyl, tetrahydronaphthyl,indanyl, idenyl, and the like. In some embodiments, aryl groups have 6carbon atoms (e.g., C₆-aryl). In some embodiments, aryl groups have fromsix to ten carbon atoms (e.g., C₆-C₁₀-aryl). In some embodiments, arylgroups have from six to sixteen carbon atoms.

The term “cycloalkyl” denotes a monovalent group derived from amonocyclic or polycyclic saturated or partially unsatured carbocyclicring compound. Examples of C₃-C₆-cycloalkyl include, but are not limitedto, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl; examples ofC₃-C₈-cycloalkyl include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; andexamples of C₃-C₁₂-cycloalkyl include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1]heptyl,and bicyclo[2.2.2]octyl. Also contemplated are monovalent groups derivedfrom a monocyclic or polycyclic carbocyclic ring compound having acarbon-carbon double bond by the removal of a single hydrogen atom.Examples of such groups include, but are not limited to, cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl,and the like.

The term “heteroaryl” refers to a mono- or poly-cyclic (e.g., bi-, ortri-cyclic or more) fused or non-fused moiety or ring system having atleast one aromatic ring, where one or more of the ring-forming atoms isa heteroatom such as oxygen, sulfur, or nitrogen. In some embodiments,the heteroaryl group has one to eight carbon atoms, one to six carbonatoms, two to 6 carbon atoms (e.g., C₁-C₈-heteroaryl, C₁-C₆-heteroaryl,or C₂-C₆-heteroaryl). In further embodiment the heteroaryl group has oneto fifteen carbon atoms. In some embodiments, the heteroaryl groupcontains five to sixteen ring atoms of which one ring atom is selectedfrom oxygen, sulfur, and nitrogen; zero, one, two, or three ring atomsare additional heteroatoms independently selected from oxygen, sulfur,and nitrogen; and the remaining ring atoms are carbon. Heteroarylincludes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl,pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl,thiazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, indolyl,quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl,acridinyl, and the like.

The term “heterocyclyl” refers to a non-aromatic 3-, 4-, 5-, 6- or7-membered ring or a bi- or tri-cyclic group fused of non-fused system,where (i) each ring contains between one and three heteroatomsindependently selected from oxygen, sulfur, and nitrogen and theremaining atoms are carbon (e.g., C₂-C₆-heterocyclyl,C₃-C₆-heterocyclyl, or C₃-C₅-heterocyclyl), (ii) each 5-membered ringhas 0 to 1 double bonds and each 6-membered ring has 0 to 2 doublebonds, (iii) the nitrogen and sulfur heteroatoms can optionally beoxidized, (iv) the nitrogen heteroatom can optionally be quaternized,and (iv) any of the above rings can be fused to a benzene ring. The term“heterocyclyl” includes, but is not limited to, [1,3]dioxolane,pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

The terms “halo” and “halogen” refer to an atom selected from fluorine,chlorine, bromine and iodine.

The term “haloalkyl” refers to alkl radicals wherein any one or more ofthe alkyl carbon atoms is substituted with halo as defined above.Haloalkyl embraces monohaloalkyl, dihaloalkyl, and polyhaloalkylradicals. The term “haloalkyl” includes, but is not limited to,fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl,dichloromethyl, trichloromethyl, and pentafluoroethyl.

The term “hydroxy” refers to an —OH radical.

The term “HDAC” refers to histone deacetylases, which are enzymes thatremove the acetyl groups from the lysine residues in core histones, thusleading to the formation of a condensed and transcriptionally silencedchromatin. There are currently 18 known histone deacetylases, which areclassified into four groups. Class I HDACs, which include HDAC1, HDAC2,HDAC3, and HDAC8, are related to the yeast RPD3 gene. Class II HDACs,which include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, are relatedto the yeast Hda1 gene. Class III HDACs, which are also known as thesirtuins are related to the Sir2 gene and include SIRT1-7. Class IVHDACs, which contains only HDAC11, has features of both Class I and IIHDACs. The term “HDAC” refers to any one or more of the 18 known histonedeacetylases, unless otherwise specified.

The term “inhibitor” is synonymous with the term antagonist.

The term “pharmaceutically acceptable salt” refers to those salts of thecompounds formed by the process of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Additionally, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present invention include the conventionalnon-toxic salts of the parent compound formed, for example, fromnon-toxic inorganic or organic acids. The pharmaceutically acceptablesalts of the present invention can be synthesized from the parentcompound, which contains a basic or acidic moiety by conventionalchemical methods. Generally, such salts can be prepared by reacting thefree acid or base forms of these compounds with a stoichiometric amountof the appropriate base or acid in water or in an organic solvent, or ina mixture of the two; generally, nonaqueous media like ether, ethylacetate, ethanol, isopropanol, or acetonitrile are preferred. Lists ofsuitable salts are found in Remington's Pharmaceutical Sciences, 17^(th)ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal ofPharmaceutical Science, 66, 2 (1977), each of which is incorporatedherein by reference in its entirety.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable” refers to compounds which possess stability sufficient toallow manufacture and which maintains the integrity of the compound fora sufficient period of time to be useful for the purposes detailedherein (e.g., therapeutic or prophylactic administration to a subject).

The term “subject” refers to a mammal. A subject therefore refers to,for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like.Preferably the subject is a human. When the subject is a human, thesubject can be referred to herein as a patient.

Compounds of the Invention

In one aspect, provided herein is a compound of Formula I:

or a pharmaceutically acceptable salt thereof,

wherein,

-   -   X¹ is CR⁷ or N;    -   X² is CH or N;    -   Y is selected from the group consisting of

-   -   Z is selected from the group consisting of H, C₁-C₆-alkyl,        C₆-aryl, C(O)NR⁴R⁵, C(O)OR⁶, C(O)C₁-C₆-alkyl,        C(O)C₀-C₆-alkyl-C₆-aryl, C(O)—C₃-C₆-cycloalkyl,        C(O)—C₂-C₆-heterocyclyl, and C(O)C₀₋₆-alkyl-heteroaryl, wherein        the aryl, heteroaryl, cycloalkyl, and heterocyclyl groups are        optionally substituted by 1 or 2 of C₁-C₆-alkyl, halo,        C₁-C₆-haloalkyl, hydroxy, or C₁-C₆-alkoxy;    -   R^(a) and R^(b) are H, or R^(a) and R^(b) together form a fused        C₆-aryl;    -   R¹ is selected from the group consisting of H and C₁-C₆-alkyl;    -   R² is selected from the group consisting of H, C₁-C₆-alkyl, and        C₆-aryl;    -   R³ is selected from the group consisting of H, C₁-C₆-alkyl, and        C₆-aryl;    -   or R² and R³ together form a C₃-C₆-heterocyclyl;    -   R⁴ is selected from the group consisting of H, C₁-C₆-alkyl,        C₁-C₆-alkyl-OH, and C₁-C₆—NH₂;    -   R⁵ is C₁-C₆-alkyl;    -   or R⁴ and R⁵ together form a C₂-C₆-heterocyclyl, wherein        heterocyclyl is optionally substituted by 1 or 2 of C₁-C₆-alkyl,        halo, C₁-C₆-haloalkyl, hydroxy, or C₁-C₆-alkoxy;    -   R⁶ is selected from the group consisting of C₁-C₆-alkyl and        C₀-C₆-alkyl-C₆-aryl, wherein aryl is optionally substituted by 1        or 2 of C₁-C₆-alkyl, halo, or hydroxy; and    -   R⁷ is selected from the group consisting of H, C₁-C₆-alkyl, and        C₃-C₆-cycloalkyl.        In an embodiment of the compound of Formula I, R^(a) and R^(b)        are H and R³ is selected from the group consisting of H and        C₆-aryl.

In an embodiment of the compound of Formula I, Y is selected from thegroup consisting of:

In another embodiment of the compound of Formula I, Z is selected fromthe group consisting of H, C₁-C₆-alkyl, C₆-aryl, C(O)OR⁶,C(O)C₁-C₆-alkyl, C(O)C₀-C₆-alkyl-C₆-aryl, C(O)—C₃-C₆-cycloalkyl, andC(O)C₀₋₆-alkyl-heteroaryl, wherein the aryl, heteroaryl, and cycloalkylgroups are optionally substituted by 1 or 2 of C₁-C₆-alkyl, halo,C₁-C₆-haloalkyl, hydroxy, or C₁-C₆-alkoxy.

In an embodiment, the compound of Formula I is a compound of Formula II:

-   -   or a pharmaceutically acceptable salt thereof.

In an embodiment of the compound of Formula I or Formula II, X¹ and X²are each N, or X¹ and X² are each CH.

In another embodiment of the compound of Formula I or Formula II, Y is:

In another embodiment of the compound of Formula I or Formula II, Z isselected from the group consisting of C(O)NR⁴R⁵, C(O)OR⁶,C(O)—C₃-C₆-cycloalkyl, C(O)—C₂-C₆-heterocyclyl, andC(O)C₀₋₆-alkyl-heteroaryl, wherein heteroaryl, cycloalkyl, orheterocyclyl are optionally substituted by 1 or 2 of C₁-C₆-alkyl, halo,or hydroxy; and

R⁶ is C₆-aryl.

In yet another embodiment of the compound of Formula I or Formula II, Zis selected from the group consisting of H, C₁-C₆-alkyl, and C₆-aryl.

In an embodiment of the compound of Formula I or Formula II, R¹ is H.

In another embodiment of the compound of Formula I or Formula II, R² isH.

In another embodiment of the compound of Formula I or Formula II, R³ isH, methyl, ethyl, isopropyl, or phenyl. In another embodiment of thecompound of Formula I or Formula II, R³ is selected from the groupconsisting of H or C₆-aryl. In yet a further embodiment of the compoundof Formula I or Formula II, R³ is H.

In an embodiment, the compound of Formula I is a compound of FormulaIIa:

-   -   or a pharmaceutically acceptable salt thereof.

In an embodiment of the compound of Formula IIa, Y is:

In another embodiment of the compound of Formula IIa, Z is selected fromthe group consisting of C(O)NR⁴R⁵, C(O)OR⁶, C(O)—C₃-C₆-cycloalkyl,C(O)—C₂-C₆-heterocyclyl, and C(O)C₀₋₆-alkyl-heteroaryl, whereinheteroaryl, cycloalkyl, or heterocyclyl are optionally substituted by 1or 2 of C₁-C₆-alkyl, halo, or hydroxy; and

R⁶ is C₆-aryl.

In yet another embodiment of the compound of Formula IIa, Z is selectedfrom the group consisting of H, C₁-C₆-alkyl, and C₆-aryl.

In an embodiment of the compound of Formula IIa, R¹ is H.

In another embodiment of the compound of Formula IIa, R² is H.

In another embodiment of the compound of Formula IIa, R³ is H, methyl,ethyl, isopropyl, or phenyl. In yet a further embodiment of the compoundof Formula IIa, R³ is H.

In a specific embodiment, R¹ is H; R² is H; and R³ is H.

In a further embodiment, the compound of Formula I is a compound ofFormula III:

or a pharmaceutically acceptable salt thereof.

In an embodiment of the compound of Formula III, X¹ and X² are each N,or X¹ and X² are each CH.

In an embodiment of the compound of Formula III, R² is H.

In an embodiment of the compound of Formula III, R³ is H, methyl, orisopropyl.

In an embodiment of the compound of Formula III, R⁴ is H and R⁵ isC₁-C₆-alkyl.

In another embodiment of the compound of Formula III, R⁴ and R⁵ togetherform a heterocyclyl selected from the group consisting of morpholinyl,piperidinyl, piperazinyl, and pyrrolidinyl, wherein the morpholinyl,piperidinyl, piperazinyl, and pyrrolidinyl are optionally substituted by1 or 2 of C₁-C₆-alkyl, halo, or hydroxy.

In another embodiment of the compound of Formula III, X¹ and X² are N;

R¹ is H;

R² is H;

R³ is H or C₁-C₄-alkyl; and

R⁴ and R⁵ together form a heterocyclyl selected from the groupconsisting of morpholinyl, piperidinyl, piperazinyl, and pyrrolidinyl,wherein the morpholinyl, piperidinyl, piperazinyl, and pyrrolidinyl areoptionally substituted by 1 or 2 of C₁-C₆-alkyl, halo, or hydroxy.

In another embodiment of the compound of Formula III, X¹ and X² are N;

R¹ is H;

R² is H;

R³ is H; and

R⁴ and R⁵ together form a heterocyclyl selected from the groupconsisting of morpholinyl, piperidinyl, piperazinyl, and pyrrolidinyl,wherein the morpholinyl, piperidinyl, piperazinyl, and pyrrolidinyl areoptionally substituted by 1 or 2 of C₁-C₆-alkyl, halo, or hydroxy.

In a further embodiment, the compound of Formula I is a compound ofFormula IIIa:

or a pharmaceutically acceptable salt thereof.

In an embodiment of the compound of Formula IIIa, R² is H.

In an embodiment of the compound of Formula IIIa, R³ is H, methyl, orisopropyl. In a further embodiment of the compound of Formula IIIa, R³is H.

In an embodiment of the compound of Formula III, R⁴ is H and R⁵ isC₁-C₆-alkyl.

In an embodiment of the compound of Formula IIIa, R⁴ and R⁵ togetherform a heterocyclyl selected from the group consisting of morpholinyl,piperidinyl, piperazinyl, and pyrrolidinyl, wherein the morpholinyl,piperidinyl, piperazinyl, and pyrrolidinyl are optionally substituted by1 or 2 of C₁-C₆-alkyl, halo, or hydroxy.

In another embodiment of the compound of Formula IIIa,

R¹ is H;

R² is H; and

R³ is H.

In another embodiment of the compound of Formula IIIa,

R¹ is H;

R² is H;

R³ is H; and

R⁴ and R⁵ together form a heterocyclyl selected from the groupconsisting of morpholinyl, piperidinyl, piperazinyl, and pyrrolidinyl,wherein the morpholinyl, piperidinyl, piperazinyl, and pyrrolidinyl areoptionally substituted by 1 or 2 of C₁-C₆-alkyl, halo, or hydroxy.

In another aspect, provided herein is a compound selected from any ofthe compounds presented in Table 1:

TABLE 1 IC50 (nM) ID Structure HDAC1 HDAC2 HDAC3 001

6.5 38 427 002

7 28 203 003

15 56 204 004

2.0 21 286 005

1.2 4.9 82 006

4.7 22 274 007

118 703 929 008

9 37 124 009

32 145 250 010

7.3 26 195 011

8.5 32 201 012

19 177 1269 013

4 14 412 014

63 299 >2000 015

3.2 12 149 016

3.2 13 145 017

18 99 1739 018

12 78 516 019

0.9 4.2 43 020

3.7 17 304 021

15 72 555 022

5 18 203 023

3.9 20 267 024

6 26 287 025

7 26 355 026

10 72 315 027

121 690 >2000 028

74 443 >2000 029

12 17 541 030

7.1 9.4 466 031

91 71 264 032

17 20 397 033

48 211 >2000 034

93 346 >2000 035

22 196 1398 036

74 996 >2000 037

432 377 1855 038

101 319 >2000 039

17 59 321 040

8 39 277 041

14 26 252 042

210 714 >2000and pharmaceutically acceptable salts thereof.

In another aspect, provided herein is a compound selected from any ofthe compounds presented in Table 1a:

TABLE 1a ID Structure ID Structure 001

002

004

005

006

007

008

009

012

013

014

015

016

017

018

019

020

021

022

023

024

025

026

027

028

030

031

032

033

034

035

036

037

038

039

040

041

042

and pharmaceutically acceptable salts thereof.

In preferred embodiments, the compounds of the instant invention haveone or more of the following properties: the compound is capable ofinhibiting at least one histone deacetylase (HDAC); the compound iscapable of inhibiting HDAC1 and/or HDAC2; the compound selectivelyinhibits HDAC1 and/or HDAC2 over other HDACs.

Another object of the present invention is the use of a compound asdescribed herein (e.g., of any formulae herein) in the manufacture of amedicament for use in the treatment of a disorder or disease herein.Another object of the present invention is the use of a compound asdescribed herein (e.g., of any formulae herein) for use in the treatmentof a disorder or disease herein.

In another aspect, provided herein is a method of synthesizing acompound of Formula I, Formula II, Formula IIa, Formula III, or FormulaIIIa, a compound presented in Table 1, a compound presented in Table 1a,or pharmaceutically acceptable salts thereof. The synthesis of thecompounds of the invention can be found in the Examples below. Anembodiment is therefore a method of making a compound of any of theformulae herein using any one, or combination of, reactions delineatedherein. The method can include the use of one or more intermediates orchemical reagents delineated herein.

In another aspect, provided herein is an isotopically labeled compoundof any of the formulae delineated herein. Such compounds have one ormore isotope atoms which can be radioactive (e.g., ³H, ²H, ¹⁴C, ¹³C,³⁵S, ³²P ¹²⁵I, and ¹³¹I) introduced into the compound. Such compoundsare useful for drug metabolism studies and diagnostics, as well astherapeutic applications.

Protected derivatives of the compounds of the invention can be made bymeans known to those of ordinary skill in the art. A detaileddescription of techniques applicable to the creation of protectinggroups and their removal can be found in T. W. Greene, “ProtectingGroups in Organic Chemistry,” 3rd edition, John Wiley and Sons, Inc.,1999, and subsequent editions thereof.

Compounds of the present invention can be conveniently prepared, orformed during the process of the invention, as solvates (e.g.,hydrates). Hydrates of compounds of the present invention can beconveniently prepared by recrystallization from an aqueous/organicsolvent mixture, using organic solvents such as dioxan, tetrahydrofuranor methanol.

In addition, compounds of this invention can have one or more doublebonds, or one or more asymmetric centers. Such compounds can occur asracemates, racemic mixtures, single enantiomers, individualdiastereomers, diastereomeric mixtures, and cis- or trans- or E- orZ-double isomeric forms, and other stereoisomeric forms that can bedefined, in terms of absolute stereochemistry, as (R)- or (S)-, or as(D)- or (L)- for amino acids. All such isomeric forms of these compoundsare expressly included in the present invention. Optical isomers can beprepared from their respective optically active precursors by theprocedures described above, or by resolving the racemic mixtures. Theresolution can be carried out in the presence of a resolving agent, bychromatography or by repeated crystallization or by some combination ofthese techniques which are known to those skilled in the art. Furtherdetails regarding resolutions can be found in Jacques, et al.,“Enantiomers, Racemates, and Resolutions” (John Wiley & Sons, 1981). Thecompounds of this invention can also be represented in multipletautomeric forms, in such instances, the invention expressly includesall tautomeric forms of the compounds described herein. When thecompounds described herein contain olefinic double bonds or othercenters of geometric asymmetry, and unless specified otherwise, it isintended that the compounds include both E and Z geometric isomers.Likewise, all tautomeric forms are also intended to be included. Theconfiguration of any carbon-carbon double bond appearing herein isselected for convenience only and is not intended to designate aparticular configuration unless the text so states; thus a carbon-carbondouble bond depicted arbitrarily herein as trans can be cis, trans, or amixture of the two in any proportion. All such isomeric forms of suchcompounds are expressly included in the present invention.

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, further methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps can beperformed in an alternate sequence or order to give the desiredcompounds. In addition, the solvents, temperatures, reaction durations,etc. delineated herein are for purposes of illustration only and one ofordinary skill in the art will recognize that variation of the reactionconditions can produce the desired compounds of the present invention.Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing the compoundsdescribed herein are known in the art.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

Pharmaceutical Compositions

Also provided herein is a pharmaceutical composition comprising acompound of the instant invention, or a pharmaceutically acceptable saltthereof, together with a pharmaceutically acceptable carrier.

In an aspect, provided herein is a pharmaceutical composition comprisingany of the compounds of the instant invention (i.e., compounds ofFormula I, Formula II, Formula IIa, Formula III, or Formula IIIa, acompound presented in Table 1, a compound presented in Table 1a, orpharmaceutically acceptable salts thereof), together with apharmaceutically acceptable carrier.

In an aspect, provided herein is a pharmaceutical composition comprisinga compound of Formula I, or a pharmaceutically acceptable salt thereof,together with a pharmaceutically acceptable carrier.

In an aspect, provided herein is a pharmaceutical composition comprisinga compound of Formula II, or a pharmaceutically acceptable salt thereof,together with a pharmaceutically acceptable carrier.

In an aspect, provided herein is a pharmaceutical composition comprisinga compound of Formula IIa, or a pharmaceutically acceptable saltthereof, together with a pharmaceutically acceptable carrier.

In an aspect, provided herein is a pharmaceutical composition comprisinga compound of Formula III, or a pharmaceutically acceptable saltthereof, together with a pharmaceutically acceptable carrier.

In an aspect, provided herein is a pharmaceutical composition comprisinga compound of Formula IIIa, or a pharmaceutically acceptable saltthereof, together with a pharmaceutically acceptable carrier.

In an aspect, provided herein is a pharmaceutical composition comprisinga compound of Table 1, or pharmaceutically acceptable salts thereof,together with a pharmaceutically acceptable carrier.

In an aspect, provided herein is a pharmaceutical composition comprisinga compound of Table 1a, or pharmaceutically acceptable salts thereof,together with a pharmaceutically acceptable carrier.

In an aspect, provided herein is a pharmaceutical composition comprisingCompound 005, or a pharmaceutically acceptable salt thereof, togetherwith a pharmaceutically acceptable carrier.

These pharmaceutical compositions comprise a therapeutically effectiveamount of a compound of the present invention formulated together withone or more pharmaceutically acceptable carriers. The term“pharmaceutically acceptable carrier” means a non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. The pharmaceutical compositions canbe administered to humans and other animals orally, rectally,parenterally, intracisternally, intravaginally, intraperitoneally,topically (as by powders, ointments, or drops), buccally, or as an oralor nasal spray.

Compounds of the invention can be administered as pharmaceuticalcompositions by any conventional route, in particular enterally, forexample, orally, e.g., in the form of tablets or capsules, orparenterally, e.g., in the form of injectable solutions or suspensions,topically, e.g., in the form of lotions, gels, ointments or creams, orin a nasal or suppository form.

Pharmaceutical compositions comprising a compound of the presentinvention in free form or in a pharmaceutically acceptable salt form inassociation with at least one pharmaceutically acceptable carrier ordiluent can be manufactured in a conventional manner by mixing,granulating or coating methods. For example, oral compositions can betablets or gelatin capsules comprising the active ingredient togetherwith a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearicacid, its magnesium or calcium salt and/or polyethyleneglycol; fortablets also c) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and or polyvinylpyrrolidone; if desired d)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or e) absorbents, colorants, flavors andsweeteners. Injectable compositions can be aqueous isotonic solutions orsuspensions, and suppositories can be prepared from fatty emulsions orsuspensions. The compositions can be sterilized and/or containadjuvants, such as preserving, stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they can also contain other therapeuticallyvaluable substances. Suitable formulations for transdermal applicationsinclude an effective amount of a compound of the present invention witha carrier. A carrier can include absorbable pharmacologically acceptablesolvents to assist passage through the skin of the host. For example,transdermal devices are in the form of a bandage comprising a backingmember, a reservoir containing the compound optionally with carriers,optionally a rate controlling barrier to deliver the compound to theskin of the host at a controlled and predetermined rate over a prolongedperiod of time, and means to secure the device to the skin. Matrixtransdermal formulations can also be used. Suitable formulations fortopical application, e.g., to the skin and eyes, are preferably aqueoussolutions, ointments, creams or gels well-known in the art. Such cancontain solubilizers, stabilizers, tonicity enhancing agents, buffersand preservatives.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings, release controlling coatings and other coatings well known inthe pharmaceutical formulating art. In such solid dosage forms theactive compound can be admixed with at least one inert diluent such assucrose, lactose or starch. Such dosage forms can also comprise, as isnormal practice, additional substances other than inert diluents, e.g.,tableting lubricants and other tableting aids such a magnesium stearateand microcrystalline cellulose. In the case of capsules, tablets andpills, the dosage forms can also comprise buffering agents.

Methods for Treating

Provided herein are methods for treating or preventing disorders in asubject, such as a human or other animal, by administering to thesubject a therapeutically effective amount of a compound of theinvention, in such amounts and for such time as is necessary to achievethe desired result. The term “therapeutically effective amount” of acompound of the invention means a sufficient amount of the compound soas to decrease the symptoms of a disorder in a subject. As is wellunderstood in the medical arts a therapeutically effective amount of acompound of this invention will be at a reasonable benefit/risk ratioapplicable to any medical treatment.

The term “treating” or “treatment” as used herein comprises relieving,reducing or alleviating at least one symptom in a subject or effecting adelay of progression of a disease. For example, treatment can be thediminishment of one or several symptoms of a disorder or completeeradication of a disorder, such as cancer. Within the meaning of thepresent disclosure, the term “treat” also denotes to arrest, delay theonset (i.e., the period prior to clinical manifestation of a disease)and/or reduce the risk of developing or worsening a disease. The term“protect” is used herein to mean prevent, delay, or treat, or all, asappropriate, development, continuance or aggravation of a disease in asubject, e.g., a mammal or human. The term “prevent”, “preventing” or“prevention” as used herein comprises the prevention of at least onesymptom associated with or caused by the state, disease or disorderbeing prevented.

In general, compounds of the invention will be administered intherapeutically effective amounts via any of the usual and acceptablemodes known in the art, either singly or in combination with one or moretherapeutic agents. A therapeutically effective amount can vary widelydepending on the severity of the disease, the age and relative health ofthe subject, the potency of the compound used and other factors.

In certain embodiments, a therapeutic amount or dose of the compounds ofthe present invention can range from about 0.1 mg/kg to about 500 mg/kg(about 0.18 mg/m² to about 900 mg/m²), alternatively from about 1 toabout 50 mg/kg (about 1.8 to about 90 mg/m²). In general, treatmentregimens according to the present invention comprise administration to apatient in need of such treatment from about 10 mg to about 1000 mg ofthe compound(s) of this invention per day in single or multiple doses.Therapeutic amounts or doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention can beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, can be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease. Thesubject can, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of thecompounds and compositions of the present invention will be decided bythe attending physician within the scope of sound medical judgment. Thespecific inhibitory dose for any particular patient will depend upon avariety of factors including the disorder being treated and the severityof the disorder; the activity of the specific compound employed; thespecific composition employed; the age, body weight, general health, sexand diet of the patient; the time of administration, route ofadministration, and rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed; and like factors well known in themedical arts.

In one aspect, the invention provides a method of selectively inhibitingthe activity of each of HDAC and/or HDAC2 over other HDACs in a subject,comprising administering a compound of Formula I, Formula II, FormulaIIa, Formula III, or Formula IIIa, a compound presented in Table 1, acompound presented in Table 1a, or pharmaceutically acceptable saltsthereof.

In an embodiment, the compound has a selectivity for each of HDAC1and/or HDAC2 of about 2 to 1000 (including ranges such as, e.g., 5 to1000, 10 to 1000, 5 to 100, etc.) fold greater than for other HDACs. Inanother embodiment, the compound has a selectivity for each of HDAC1and/or HDAC2 when tested in a HDAC enzyme assay of about 2 to 1000(including ranges such as, e.g., 5 to 1000, 10 to 1000, 5 to 100, etc.)fold greater than for other HDACs.

In another aspect, the invention provides a method for treating adisease mediated by an HDAC, specifically HDAC1 and/or HDAC2 in asubject comprising administering to the subject a compound of Formula I,Formula II, Formula IIa, Formula III, or Formula IIIa, a compoundpresented in Table 1, a compound presented in Table 1a, orpharmaceutically acceptable salts thereof. The selective HDAC1 and HDAC2inhibitors of the present invention have favorable pharmacokineticprofiles (see, e.g., Example 44).

In an aspect, provided herein is a method for treating a diseasemediated by HDAC1 and/or HDAC2 in a subject comprising administering tothe subject a therapeutically effective amount of compound of Formula I,or a pharmaceutically acceptable salt thereof.

In an aspect, provided herein is a method for treating a diseasemediated by HDAC1 and/or HDAC2 in a subject comprising administering tothe subject a therapeutically effective amount of compound of FormulaII, or a pharmaceutically acceptable salt thereof.

In an aspect, provided herein is a method for treating a diseasemediated by HDAC1 and/or HDAC2 in a subject comprising administering tothe subject a therapeutically effective amount of compound of FormulaIIa, or a pharmaceutically acceptable salt thereof.

In an aspect, provided herein is a method for treating a diseasemediated by HDAC1 and/or HDAC2 in a subject comprising administering tothe subject a therapeutically effective amount of compound of FormulaIII, or a pharmaceutically acceptable salt thereof.

In an aspect, provided herein is a method for treating a diseasemediated by HDAC1 and/or HDAC2 in a subject comprising administering tothe subject a therapeutically effective amount of compound of FormulaIIIa, or a pharmaceutically acceptable salt thereof.

In an aspect, provided herein is a method for treating a diseasemediated by HDAC1 and/or HDAC2 in a subject comprising administering tothe subject a therapeutically effective amount of compound of Table 1,or pharmaceutically acceptable salts thereof.

In an aspect, provided herein is a method for treating a diseasemediated by HDAC1 and/or HDAC2 in a subject comprising administering tothe subject a therapeutically effective amount of compound of Table 1a,or pharmaceutically acceptable salts thereof.

In an aspect, provided herein is a method for treating a diseasemediated by HDAC1 and/or HDAC2 in a subject comprising administering tothe subject a therapeutically effective amount of Compound 005, or apharmaceutically acceptable salt thereof.

Inhibition of HDAC1 and HDAC2 is sufficient to derepress fetal globin.In cultured human CD34+ bone marrow cells undergoing erythroiddifferentiation, these compounds can induce a dose dependent increase infetal hemoglobin expression (see, e.g., Example 45).

Thus, the compounds are capable of derepressing fetal globin throughHDAC inhibition. Accordingly, in an embodiment, the compounds are ableto treat a subject suffering from or susceptible to a hemoglobinopathy.In a preferred embodiment, the compounds are able to treat sickle-celldisease or beta-thalessemia.

In another embodiment, the compounds of the invention are useful in thetreatment of myelodysplastic syndromes.

In certain embodiments, the compounds of the present invention areuseful as anti-cancer agents. The compounds of the invention are capableof inducing apoptosis in cancer cells thereby able to treat a diseasesuch as a cancer or proliferation disease. In an embodiment, thecompound of the invention can be useful in the treatment of cancer, byeffecting tumor cell death or inhibiting the growth of tumor cells.

In certain embodiments, the cancer is lung cancer, colon and rectalcancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer,brain cancer, kidney cancer, ovarian cancer, stomach cancer, skincancer, bone cancer, gastric cancer, breast cancer, glioma,glioblastoma, neuroblastom, hepatocellular carcinoma, papillary renalcarcinoma, head and neck squamous cell carcinoma, leukemia, lymphomas,myelomas, retinoblastoma, cervical cancer, melanoma and/or skin cancer,bladder cancer, uterine cancer, testicular cancer, esophageal cancer,and solid tumors. In some embodiments, the cancer is lung cancer, coloncancer, breast cancer, neuroblastoma, leukemia, or lymphomas. In otherembodiments, the cancer is lung cancer, colon cancer, breast cancer,neuroblastoma, leukemia, or lymphoma. In a further embodiment, thecancer is non-small cell lung cancer (NSCLC) or small cell lung cancer.In another embodiment, the cancer is neuroblastoma.

In further embodiments, the cancer is a hematologic cancer, such asleukemia or lymphoma. In a certain embodiment, lymphoma is Hodgkinslymphoma or Non Hodgkin's lymphoma. In certain embodiments, leukemia ismyeloid, lymphocytic, myelocytic, lymphoblastic, or megakaryoticleukemia. In a particular embodiment, the leukemia is acute myelogenousleukemia and megakaryocytic leukemia.

In another aspect, provided herein is a method for treating sickle celldisease, beta thalassemia, myelodysplastic syndrome, acute myelogenousleukemia, neuroblastoma, or megakaryocytic leukemia in a subjectcomprising administering to the subject in need thereof atherapeutically effective amount of a compound of Formula I, Formula II,Formula IIa, Formula III, or Formula IIIa, a compound presented in Table1, a compound presented in Table 1a, or pharmaceutically acceptablesalts thereof.

In an aspect, provided herein is a method for treating sickle celldisease, beta thalassemia, myelodysplastic syndrome, acute myelogenousleukemia, neuroblastoma, or megakaryocytic leukemia in a subjectcomprising administering to the subject a therapeutically effectiveamount of compound of Formula I, or a pharmaceutically acceptable saltthereof.

In an aspect, provided herein is a method for treating sickle celldisease, beta thalassemia, myelodysplastic syndrome, acute myelogenousleukemia, neuroblastoma, or megakaryocytic leukemia in a subjectcomprising administering to the subject a therapeutically effectiveamount of compound of Formula II, or a pharmaceutically acceptable saltthereof.

In an aspect, provided herein is a method for treating sickle celldisease, beta thalassemia, myelodysplastic syndrome, acute myelogenousleukemia, neuroblastoma, or megakaryocytic leukemia in a subjectcomprising administering to the subject a therapeutically effectiveamount of compound of Formula IIa, or a pharmaceutically acceptable saltthereof.

In an aspect, provided herein is a method for treating sickle celldisease, beta thalassemia, myelodysplastic syndrome, acute myelogenousleukemia, neuroblastoma, or megakaryocytic leukemia in a subjectcomprising administering to the subject a therapeutically effectiveamount of compound of Formula III, or a pharmaceutically acceptable saltthereof.

In an aspect, provided herein is a method for treating sickle celldisease, beta thalassemia, myelodysplastic syndrome, acute myelogenousleukemia, neuroblastoma, or megakaryocytic leukemia in a subjectcomprising administering to the subject a therapeutically effectiveamount of compound of Formula IIIa, or a pharmaceutically acceptablesalt thereof.

Methods delineated herein include those wherein the subject isidentified as in need of a particular stated treatment. Identifying asubject in need of such treatment can be in the judgment of a subject ora health care professional and can be subjective (e.g. opinion) orobjective (e.g. measurable by a test or diagnostic method).

Also, as discussed above, the compounds of the invention are selectiveinhibitors of HDAC1 and/or HDAC2 and, as such, are useful in thetreatment of disorders modulated by these histone deacetylases (HDACs).For example, compounds of the invention can be useful in the treatmentof cancer (e.g., lung cancer, colon cancer, breast cancer,neuroblastoma, leukemia, or lymphomas, etc.). Accordingly, in yetanother aspect, according to the methods for treatment of the presentinvention, tumor cells are killed, or their growth is inhibited bycontacting said tumor cells with an inventive compound or composition,as described herein.

Thus, in another aspect of the invention, methods for the treatment ofcancer are provided comprising administering a therapeutically effectiveamount of an inventive compound (i.e., of any of the formulae herein),as described herein, to a subject in need thereof. In certainembodiments, the subject is identified as in need of such treatment. Incertain embodiments, a method for the treatment of cancer is providedcomprising administering a therapeutically effective amount of aninventive compound, or a pharmaceutical composition comprising aninventive compound to a subject in need thereof, in such amounts and forsuch time as is necessary to achieve the desired result. In certainembodiments of the present invention a “therapeutically effectiveamount” of the inventive compound or pharmaceutical composition is thatamount effective for killing or inhibiting the growth of tumor cells.The compounds and compositions, according to the method of the presentinvention, can be administered using any amount and any route ofadministration effective for killing or inhibiting the growth of tumorcells. Thus, the expression “amount effective to kill or inhibit thegrowth of tumor cells,” as used herein, refers to a sufficient amount ofagent to kill or inhibit the growth of tumor cells. The exact amountrequired will vary from subject to subject, depending on the species,age, and general condition of the subject, the severity of theinfection, the particular anticancer agent, its mode of administration,and the like.

In certain embodiments, the method involves the administration of atherapeutically effective amount of the compound or a pharmaceuticallyacceptable derivative thereof to a subject (including, but not limitedto a human or animal) in need of it. In certain embodiments, theinventive compounds as useful for the treatment of cancer and otherproliferative disorders including, but not limited to lung cancer (e.g.non-small cell lung cancer), colon and rectal cancer, breast cancer,prostate cancer, liver cancer, pancreatic cancer, brain cancer, kidneycancer, ovarian cancer, stomach cancer, skin cancer, bone cancer,gastric cancer, breast cancer, glioma, glioblastoma, neuroblastoma,hepatocellular carcinoma, papillary renal carcinoma, head and necksquamous cell carcinoma, leukemia (e.g., CML, AML, CLL, ALL), lymphomas(non-Hodgkin's and Hodgkin's), myelomas, retinoblastoma, cervicalcancer, melanoma and/or skin cancer, bladder cancer, uterine cancer,testicular cancer, esophageal cancer, and solid tumors.

In certain embodiments, the invention provides a method for treating ofany of the disorders described herein, wherein the subject is a human.

In accordance with the foregoing, the present invention further providesa method for preventing or treating any of the diseases or disordersdescribed above in a subject in need of such treatment, which methodcomprises administering to said subject a therapeutically effectiveamount of a compound of the invention or a pharmaceutically acceptablesalt thereof. For any of the above uses, the required dosage will varydepending on the mode of administration, the particular condition to betreated and the effect desired.

EXAMPLES

Examples have been set forth below for the purpose of illustration andto describe certain specific embodiments of the invention. However, thescope of the claims is not to be in any way limited by the examples setforth herein. Various changes and modifications to the disclosedembodiments will be apparent to those skilled in the art and suchchanges and modifications including, without limitation, those relatingto the chemical structures, substituents, derivatives, formulationsand/or methods of the invention can be made without departing from thespirit of the invention and the scope of the appended claims.Definitions of the variables in the structures in the schemes herein arecommensurate with those of corresponding positions in the formulaepresented herein.

Example 1: Synthesis of Compound 001

Step 1: Lithium bis(trimethylsilyl)amide (1.0 M solution in THF, 240 ml,240 mmol) was slowly added into a round-bottomed flask with compound 1(25 g, 120 mmol) at −76° C. under N₂. The reaction was stirred for 4 hat −76° C., and then iodomethane (15 ml, 240 mmol) was injected into thesystem. The reaction mixture was stirred at −76° C. for 30 min. and thenwarmed to room temperature and stirred overnight. The reaction mixturewas quenched with 150 ml saturated aqueous NH₄Cl, diluted with water andextracted with EtOAc (ethyl acetate, or EA). The organic layers werewashed with water and brine and dried over sodium sulfate, filtered andconcentrated to yield target compound 2 (25 g, 93%) as a light yellowsolid.

Step 2: K₂CO₃ (31 g, 224 mmol) was added into a solution of compound 2(25 g, 111 mmol) in DMSO (120 ml). H₂O₂ (100 ml) was slowly added to thesystem at 60° C., and the reaction was stirred overnight at 60° C. Thesystem was then introduced into cold water and extracted with EA. Theorganic layers were washed with water and brine and dried over sodiumsulfate, filtered and concentrated to yield target compound 3 (26 g,96%) as a white solid.

Step 3: Compound 3 (26 g, 107 mmol) was dissolved with ACN(acetonitrile) (200 ml) and 5N KOH (100 ml).1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione (15 g, 54 mmol) was thenadded into the system. The mixture was stirred overnight andconcentrated to remove the ACN. The pH of the water phase was adjustedto about 5 with 2N HCl in an ice bath, extracted with EA and separated.The pH of water phase was then adjusted to 10. The precipitate wascollected to yield compound 4 as a white solid (16 g, 69%).

Step 4: A solution of compound 4 (2 g, 9.34 mmol), 2-chloropyrimidine(2.6 g, 14.02 mmol) and DIPEA (5.3 g, 28.03 mmol) in 1,4-dioxane (25 ml)was heated at 95° C. overnight. The reaction mixture was thenconcentrated and purified by silica gel column with EA/PE=1/5 to obtaincompound 5 (1.8 g, 53%) as a light yellow solid.

Step 5: A solution of compound 5 (465 mg, 1.28 mmol), 2N NaOH (10 ml, 20mmol) in THF (10 ml), and EtOH (10 ml) was heated at 55° C. for 2 h. Thereaction solution was concentrated and he pH of the water phase wasadjusted to between about 5-6. The resulting solution was extracted withEA, the organic layers were washed with water and brine then dried oversodium sulfate, filtered and concentrated to get target compound 6 (400mg, 93%) as a white solid.

Step 6: A mixture of compound 6 (400 mg, 1.19 mmol), amine (345 mg, 1.19mmol), EDCI (307 mg, 2.38 mmol) and DMAP (290 mg, 2.38 mmol) in DMF (10mL) was heated at 55° C. overnight. The mixture was mixed with water andextracted with EA. The organic layers were washed with water and brinethen dried over sodium sulfate, filtered and concentrated. The resultingcomposition was purified by silica gel column with EA/PE=1/2 to yieldcompound 7 (400 mg, 55%) as a purple solid.

Step 7: A solution of compound 7 (400 mg, 0.65 mmol) withHCl/1,4-dioxane (5 ml, 20 mmol) in 1,4-dioxane (10 ml) was stirred atroom temperature overnight. The reaction solution was concentrated andwashed with PE to yield target compound 8 (350 mg, 100%) as a graysolid.

Step 8: Compound 8 (162 mg, 0.4 mmol) and Et3N (80 mg, 0.8 mmol) weredissolved in THF (5 ml). Isoproyl Isocyanate (CAS: 1795-48-8, 1.2 eq)was added into the system. The mixture was stirred at room temperaturefor 2 h. Then the mixture was concentrated and purified by Pre-HPLC toyield Compound 001 (35 mg, 16%). ¹H NMR (500 MHz, DMSO) δ 9.51 (s, 1H),8.84 (s, 2H), 7.50 (s, 1H), 7.44 (d, J=2.0 Hz, 1H), 7.35 (d, J=5.1 Hz,1H), 7.29 (dd, J=8.3, 2.1 Hz, 1H), 7.23 (d, J=2.7 Hz, 1H), 7.05 (dd,J=5.0, 3.6 Hz, 1H), 6.79 (d, J=8.3 Hz, 1H), 6.09 (d, J=7.6 Hz, 1H), 5.20(s, 2H), 3.53 (d, J=13.6 Hz, 2H), 3.05 (t, J=10.7 Hz, 2H), 2.25 (d,J=13.8 Hz, 2H), 1.53-1.44 (m, 2H), 1.42 (s, 3H), 1.08 (d, J=6.6 Hz, 1H),1.04 (d, J=6.6 Hz, 6H). LCMS: m/z=494 (M+H).

Example 2: Synthesis of Compound 002

Step 1: To a solution of 1 (3 g, 14.28 mmol) in a boiling flask-3-neckflushed with N₂ was added LHDMS (1M, 21.4 ml) at −78° C. The reactionwas stirred for 3 h and then 2-iodopropane (3.6 g, 21.43 mmol) wasslowly added. The reaction solution was stirred at −78° C., and warmedto room temperature overnight. The mixture was quenched with H₂O (2 ml)and concentrated, dissolved in EA (200 ml), and washed with water (100ml*2) followed by saturated NaCl (aqueous, 100 ml). The organic layerwas concentrated to obtain compound 2 as a brown solid (4 g, 100%).

Step 2: To a solution of 2 (1 g, 3.97 mmol) in DMSO (30 ml) was addedK₂CO₃ (1.6 g, 11.9 mmol). The resulting reaction mixture was stirred at60° C. H₂O₂ (30% aq, 5 ml) was then added dropwise and stirred for 2 h.EA (100 ml) was added to the mixture which was then washed with water(50 ml*2) and saturated NaCl (aq, 50 ml). Drying with anhydrous Na₂SO₄and concentrating yielded compound 3 as a white solid (1 g, 90%).

Step 3: To a solution of compound 3 (2.7 g, 10 mmol) in ACN (50 ml) wasadded KOH (4 N, aq, 50 ml) and DBDMH (2.81 g, 5 mmol) at 0° C. Theresulting reaction mixture was stirred at room temperature overnight.The mixture was concentrated and 1N HCl was added to adjust the pH toabout 6. The mixture was then extracted with EA (50 ml), and the waterphase was adjust to about pH 9 by adding KOH. The resulting water phasewas extracted with EA (50 ml×3). The EA phase was dried with anhydrousNa₂SO₄ and concentrated to yield compound 4 as a colorless liquid (1 g,40%).

Step 4: A solution of compound 4 (500 mg, 2.06 mmol) and ethyl2-chloropyrimidine-5-carboxylate (384 mg, 2.06 mmol) in NMP (10 ml) wasflushed with N₂ and stirred at 140° C. for 1 hour. EA (100 ml) was addedto the mixture, which was then washed with water (50 ml×2) and saturatedNaCl (aq, 50 ml). Concentration and purification by silica gelchromatography column (PE/EA=5/1) yielded compound 5 as a white solid(120 mg 15%).

Step 5: To a mixture of compound 5 (400 mg) in EtOH (aq, 95%, 5 ml) wasadded NaOH (2M, 5 ml) and the reaction was stirred at 55° C. for 2hours. Water (50 ml) was added to the mixture and the pH was adjusted toabout 7 with citric acid. The resulting aqeuous mixture was extractedwith EA (50 ml×3). The EA phase was dried with anhydrous Na₂SO₄ andconcentrated to yield compound 6 as a white solid (340 mg, crude).

Step 6: A solution of 6 (100 mg, crude), amine (79.6 mg, 0.274 mmol),EDCI (71 mg, 0.549 mmol), HOAT (75 mg, 0.549 mmol), DMAP (3.4 mg, 0.027mmol), DIPEA (142 mg, 1.1 mmol) in DMF (5 ml) was stirred at 55° C.overnight. EA (100 ml) was added to the mixture which was then washedwith water (50 ml×3) and concentrated to yield compound 7 as a brown oil(200 mg, crude).

Step 7: To a solution of 7 (200 mg, crude) in DCM (2 ml) was added TFA(2 ml) and the resulting mixture as stirred at rt for 2 hours. Themixture was concentrated to yield compound 8 as a brown oil (200 mg,20%).

Step 8: To a solution of compound 8 (100 mg, crude) in DCM (5 ml) wasadded DIPEA (88.8 mg, 0.688 mmol) and (44.6 mg, 0.275 mmol). Thereaction was stirred at 0° C. for 1 hour. The mixture was concentratedand purified by Pre-HPLC to yield Compound 002 as a white solid (34 mg,35%). ¹H NMR (400 MHz, DMSO) δ 9.75 (s, 1H), 9.64 (s, 1H), 8.84 (s, 2H),7.58 (s, 1H), 7.47 (d, J=1.9 Hz, 1H), 7.39 (d, J=4.9 Hz, 1H), 7.35 (dd,J=8.3, 2.1 Hz, 1H), 7.28 (d, J=3.3 Hz, 1H), 7.09-7.04 (m, 1H), 6.88 (d,J=8.4 Hz, 1H), 3.64 (d, J=10.5 Hz, 2H), 3.50 (d, J=11.9 Hz, 2H), 3.37(d, J=8.4 Hz, 2H), 2.98 (dd, J=30.8, 11.3 Hz, 6H), 2.81 (s, 3H),2.61-2.55 (m, 1H), 2.38 (d, J=11.7 Hz, 2H), 1.51 (t, J=11.1 Hz, 2H),0.86 (d, J=6.9 Hz, 6H). LCMS: m/z=563 (M+H).

Example 3: Synthesis of Compound 003

Step 1: A mixture of 2-Cl-pyrimidine (1.86 g, 10 mmol), an amine (3.00,15 mmol), and NEt3 (3.0 g, 30 mmol) in 1,4-dioxane (20 ml) was stirredat 95° C. overnight. The mixture was concentrated, EA (60 ml) andaqueous citric acid (60 mL) were added, and the resulting mixture wasstirred for 30 min. The organic layer was separated, dried, andconcentrated to yield compound 2 (3.4 g, yield: 97%) as a light yellowsolid.

Step 2: A mixture of compound 2 (3.5 g, 10 mmol) and NaOH (2M, 15 ml) inEtOH (15 ml) and THE (15 ml) was stirred at 60° C. for 2 hours. Themixture was concentrated, and aqueous citric acid was added until thepH<7. The resulting mixture was stirred for 30 min and filtered to yieldcompound 3 (2.8 g, yield: 90%) as a light yellow solid.

Step 3: A mixture of compound 3 (3.2 g, 10 mmol), compound amine (2.9 g,10 mmol), HOAT (2.0 g, 15 mmol), EDCI (3.8 g, 20 mmol) in DMF (25 ml)was stirred at 60° C. overnight. EA (100 ml) and aqueous saturated NaCl(100 ml) were added to the mixture and the resulting mixture was stirredfor 30 min. The organic layer was separated, washed by aqueous saturatedNaCl (50 ml×2), dried and concentrated to produce a residue, which waswashed by CH₃CN (10-20 ml) to yield compound 4 (2.9 g, 50%) as a graysolid.

Step 4: To a solution of compound 4 (2.9 g, 5 mmol) in DCM (30 ml) wasadded TFA (5 ml) at rt for 2 hours. The mixture was concentrated toyield compound 5 (2.9 g, 100%) without further purification.

Step 5: To a solution of compound 4 (197 mg, 0.5 mmol) and NEt3 (250 mg,2.5 mmol) in DCM (5 ml) was added morpholine-4-carbonyl chloride (194mg, 0.65 mmol) at 0° C. LCMS was used to monitor the reaction tocompletion. NH₃—H₂O (0.5 ml) was added to the reaction mixture which wasthen stirred for 30 min and concentrated to a residue. Purification bysilica gel column yielded Compound 003 (114 mg, 45%) as a light yellowsolid. H NMR (500 MHz, DMSO) δ 9.54 (s, 1H), 8.85 (s, 2H), 7.88 (d,J=7.9 Hz, 1H), 7.44 (d, J=1.9 Hz, 1H), 7.35 (d, J=5.0 Hz, 1H), 7.29 (dd,J=8.3, 2.1 Hz, 1H), 7.24 (d, J=3.4 Hz, 1H), 7.05 (dd, J=4.9, 3.7 Hz,1H), 6.79 (d, J=8.3 Hz, 1H), 5.21 (s, 2H), 4.01 (dd, J=7.2, 3.3 Hz, 1H),3.68-3.51 (m, 7H), 3.18-3.04 (m, 5H), 2.88 (t, J=11.7 Hz, 2H), 1.86 (d,J=10.0 Hz, 2H), 1.45 (dd, J=20.5, 11.3 Hz, 2H). LCMS: m/z=508 (M+H)

Example 4: Synthesis of Compound 004

Step 1: A solution of amine 1 (1 g, 4.67 mmol), methyl 4-bromobenzoate(1 g, 4.67 mmol), Pd₂(dba)₃ (428 mg, 0.47 mmol), Ruphos (218 mg, 0.47mmol), Cs₂CO₃ (4.5 g, 14.0 mmol) in Tol (50 ml) was flushed with N₂ andstirred at 98° C. overnight. The mixture was filtered, concentrated, andpurified by silica gel chromatography (PE:EA=5:1-1:1) to yield compound2 as a yellow solid (1.1 g, 65%)

Step 2: To a mixture of compound 2 (1.1 g) in EtOH (aqueous 95%, 5 ml)was added NaOH (2M, 5 ml) and the resulting solution was stirred at 55°C. for 2 hours. Water (50 ml) was added to the mixture and the pH wasadjusted to 7 with citric acid. The resulting mixture was extracted withEA (50 ml×3). The EA phase was dried with anhydrous Na₂SO₄ andconcentrated to yield compound 3 as a white solid (1 g, crude).

Step 3: A solution of compound 3 (800 mg, crude), amine (694 mg, 2.395mmol), EDCI (618 mg, 4.790 mmol), HOAT (651 mg, 4.790 mmol), DMAP (29mg, 0.239 mmol), and DIPEA (927 mg, 7.186 mmol) in DMF (20 ml) wasstirred at 55° C. for 3 days. EA (100 ml) was added to the mixture andthe resulting solution was washed with water (50 ml×3), concentrated,and purified by silica gel chromatography (PE:EA=6:1-1:1) to yieldcompound 4 as a brown solid (650 mg, 45%).

Step 4: To a solution of compound 4 (300 mg) in DCM (2 ml) was added TFA(2 ml) and the resulting solution was stirred at rt for 2 hours. Themixture was concentrated to yield compound 5 as a brown oil (400 mg,crude).

Step 5: To a solution of compound 5 (200 mg, crude) in DCM (20 ml) wasadded DIPEA (190 mg, 1.478 mmol) and morpholine-4-carbonyl chloride (110mg, 0.739 mmol). The resulting reaction mixture was stirred at 0° C. for1 hour. The mixture was concentrated and purified by Pre-HPLC to yieldCompound 004 as a white solid (67.4 mg). ¹H NMR (500 MHz, DMSO) δ 9.85(s, 1H), 7.81 (t, J=21.9 Hz, 2H), 7.58 (d, J=1.7 Hz, 1H), 7.53-7.42 (m,2H), 7.40 (d, J=3.1 Hz, 1H), 7.11 (dd, J=5.0, 3.6 Hz, 2H), 6.92 (s, 2H),3.63-3.47 (m, 4H), 3.31-3.01 (m, 8H), 1.98 (d, J=14.0 Hz, 2H), 1.63 (d,J=12.9 Hz, 2H), 1.37 (s, 3H). LCMS: m/z=520 (M+H).

Example 5: Synthesis of Compound 005

Step 1: To a solution of compound 1 (1 g, 5 mmol) and methyl4-bromobenzoate (1.1 g, 5 mmol) in toluene (20 ml) was added Pd2(dba)3(230 mg, 0.25 mmol), Ruphos (290 mg, 0.5 mmol) and Cs2CO3 (4.9 g, 15mmol). The reaction was stirred at 95° C. overnight under a N₂atmosphere. After the starting material was fully consumed, theheterogeneous mixture was filtered through diatomite and concentrated invacuo to yield a viscous oil, which was purified by silica gel column toyield compound 2 (1 g, 60%).

Step 2: Compound 2 (1 g, 3 mmol) was dissolved with MeOH (10 ml) and THE(10 ml). 2N NaOH (15 ml) was then added into the solution. The reactionwas stirred at 55° C. for 1 hour. The reaction mixture was concentratedto remove the solvent and the pH was adjusted to between about 4-5 andextracted with EA. The organic phase was washed with brine and driedover Na₂SO₄. Concentration of the organic phase yielded compound 3 (800mg, 82%) as a white solid.

Step 3: To a solution of compound 3 (500 mg, 1.56 mmol) and amine (453mg, 1.56 mmol) in DMF (10 ml) was added HOAT (421 mg, 3.1 mmol), EDCI(592 mg, 3.1 mmol) and DIPEA (400 mg, 3.1 mmol). The reaction wasstirred at 55° C. overnight. The reaction was quenched with water andextracted with EA. The organic phase was washed with brine and driedover Na₂SO₄. Purification by silica gel column yielded compound 4 (800mg, 80%) as a light yellow solid.

Step 4: To a solution of the compound 4 (800 mg, 1.35 mmol) in DCM (10ml) was added TFA (5 ml). The solution was stirred at rt for 30 min.Concentration of the solution yielded compound 5 (600 mg, 100%) as agray solid.

Step 5: To a solution of compound 5 (80 mg, 0.20 mmol) and4-methylpiperazine-1-carbonyl chloride hydrochloride (40 mg, 0.20 mmol)was added triethylamine (100 mg, 1 mmol) at 0° C. The reaction wasstirred at 0° C. for 2 h and then filtered through silica gel.

Concentration and purification by Pre-HPLC yielded Compound 004 (15 mg,15%) as a white solid. ¹H NMR (400 MHz, DMSO) δ 9.37 (s, 1H), 7.78 (d,J=8.7 Hz, 2H), 7.45 (d, J=2.1 Hz, 1H), 7.36 (dd, J=5.1, 1.0 Hz, 1H),7.28 (d, J=2.1 Hz, 1H), 7.26 (d, J=2.2 Hz, 1H), 7.24 (dd, J=3.5, 1.1 Hz,1H), 7.05 (dd, J=5.1, 3.6 Hz, 1H), 6.80 (d, J=8.3 Hz, 1H), 6.65 (d,J=8.8 Hz, 2H), 6.20 (d, J=8.1 Hz, 1H), 5.08 (s, 2H), 3.57 (d, J=13.0 Hz,3H), 3.15 (s, 4H), 2.91 (t, J=11.3 Hz, 2H), 2.33 (s, 3H), 2.20 (s, 3H),1.91 (d, J=10.2 Hz, 2H), 1.41-1.24 (m, 2H). LCMS: m/z=519 (M+H).

Example 6: Synthesis of Compound 006

Steps 1-4: Refer to steps 1-4 of Example 5 to obtain compound 5.

Step 5: To a solution of compound 5 (100 mg, 0.25 mmol) andpyrrolidine-1-carbonyl chloride (34 mg, 0.25 mmol) was addedtriethylamine (100 mg, 1 mmol) at 0° C. The reaction was stirred at 0°C. for 2 h and then filtered through silica gel. Concentration andpurification by Pre-HPLC yielded Compound 006 (10 mg, 8%) as a whitesolid. ¹H NMR (400 MHz, DMSO) δ 9.50 (s, 1H), 7.79 (d, J=8.8 Hz, 2H),7.50 (d, J=2.1 Hz, 1H), 7.40 (d, J=4.2 Hz, 1H), 7.33 (dd, J=8.3, 2.1 Hz,1H), 7.29 (d, J=2.7 Hz, 1H), 7.07 (dd, J=5.1, 3.6 Hz, 1H), 6.91 (d,J=8.3 Hz, 1H), 6.66 (d, J=8.8 Hz, 2H), 3.63 (d, J=13.2 Hz, 2H), 3.53 (s,1H), 3.26 (t, J=6.5 Hz, 4H), 2.87 (t, J=11.2 Hz, 2H), 1.91 (d, J=10.8Hz, 2H), 1.75 (t, J=6.5 Hz, 5H), 1.34 (dd, J=20.6, 10.1 Hz, 2H). LCMS:m/z=490 (M+H).

Example 7: Synthesis of Compound 007

Step 1: A solution of lithium bis(trimethylsilyl)amide (1.0 M solutionin THF, 240 ml, 240 mmol) was added slowly into a round-bottomed flaskwith compound 1 (25 g, 120 mmol) at −76° C. under nitrogen atmosphere.The mixture was stirred for 4 h at −76° C. Then iodomethane (15 ml, 240mmol) was added into the system. The reaction mixture was stirred at−76° C. for 30 min, and then warmed to room temperature and stirredovernight. The reaction mixture was quenched with 150 ml saturatedaqueous NH4Cl, diluted with water and extracted with EtOAc. The organiclayers were washed with water and brine then dried over sodium sulfate,filtered and concentrated to afford target compound 2 (25 g, 93%) as alight yellow solid.

Step 2: Added K2CO3 (31 g, 224 mmol) into the solution of the compound 2(25 g, 111 mmol) in DMSO (120 ml). H₂O₂ (100 ml) was added into thesystem at 60° C. slowly and the reaction was stirred overnight at 60° C.After completed, the system was quenched with cold water and extractedwith EA. The organic layers were washed with water and brine then driedover sodium sulfate, filtered and concentrated to get target compound 3(26 g, 96%) as a white solid.

Step 3: To a solution of compound 3 (26 g, 107 mmol) in 200 ml CH3CN wasadded 5N KOH (100 ml). Then1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione (15 g, 54 mmol) wasadded into the system. The reaction was stirred overnight. Aftercompletion, the mixture was concentrated to remove the CH3CN, and the pHof the water phase was adjusted to 5 with 2N HCl in an ice bath,extracted with EA and separated. Then the pH of water phase was adjustedto 10. The precipitate was collected to afford compound 4 (16.1 g, 69%)as a white solid.

Step 4: To a solution of compound 4 (2 g, 9.34 mmol) in 1,4-dioxane (25ml) was added ethyl 2-chloropyrimidine-5-carboxylate (2.6 g, 14.02 mmol)and DIPEA (5.3 g, 28.03 mmol). The reaction was stirred at 95° C.overnight. Concentration and purification by silica gel column withEA/PE=1/5 afforded compound 5 (1.8 g, 53%) as a light yellow solid.

Step 5: A solution of the compound 5 (465 mg, 1.28 mmol) and 2N NaOH (10ml, 20 mmol) in THE (10 ml) and EtOH (10 ml) was stirred at 55° C. for 2h. Concentration and adjustment of the pH of the water phase to 5-6 wasfollowed by extraction with EA (2*15 ml). The organic layers were washedwith water and brine then dried over sodium sulfate, filtered andconcentrated to afford the compound 6 (400 mg, 93%) as a white solid.

Step 6: A solution of the compound 6 (400 mg, 1.19 mmol), tert-butyl2-amino-4-(thiophen-2-yl)phenylcarbamate (345 mg, 1.19 mmol), EDCI (307mg, 2.38 mmol) and DMAP (290 mg, 2.38 mmol) in DMF (10 ml) was formed.The reaction was stirred at 55° C. overnight. After completion, themixture with was added into water and extracted with EA (2*15 ml). Theorganic layers were washed with water and brine then dried over sodiumsulfate, filtered and concentrated. Then purification by silica gelcolumn with EA/PE=1/2 yielded compound 7 (400 mg, 55%) as a purplesolid.

Step 7: To a solution of the compound 7 (400 mg, 0.65 mmol) in1,4-dioxane (10 ml) was added HCl/1,4-dioxane (5 ml, 20 mmol) at roomtemperature overnight. Concentration and washing with PE yielded thecompound 8 (350 mg, 100%) as a gray solid.

Step 8: To a solution of compound 8 (200 mg, 0.45 mmol) and Et3N (101mg, 2.2 eq) in THF (10 ml) was added phenyl carbonochloridate (78 mg,0.5 mmol). The mixture was stirred at room temperature for 2 h. Aftercompletion, the mixture was concentrated and purified by Prep-HPLC toafford Compound 007 (66 mg, 28%). ¹H NMR (500 MHz, DMSO) δ 9.71 (s, 1H),8.88 (s, 2H), 7.68 (s, 1H), 7.50 (s, 1H), 7.42-7.35 (m, 4H), 7.30 (d,J=2.9 Hz, 1H), 7.22 (t, J=7.3 Hz, 1H), 7.12 (d, J=7.9 Hz, 2H), 7.10-7.06(m, 1H), 6.91 (d, J=8.2 Hz, 1H), 3.76 (d, J=60.0 Hz, 2H), 3.32 (d,J=79.9 Hz, 2H), 2.40 (s, 2H), 1.65 (s, 2H), 1.48 (s, 3H). LCMS: m/z=529(M+H)⁺.

Example 8: Synthesis of Compound 008

Steps 1-7: Refer to steps 1-7 of Example 7 to obtain compound 8.

Step 8: To a solution of compound 8 (100 mg, 0.22 mmol) and Et3N (44 mg,0.44 mmol) in THE (5 ml) was added morphine-1-carbonyl chloride (36 mg,1.1 eq). The mixture was stirred at room temperature for 2 hours. Aftercompletion, the mixture was concentrated and purified by Prep-HPLC toafford Compound 008 (50 mg, 44%). ¹H NMR (500 MHz, DMSO) δ 9.52 (s, 1H),8.85 (s, 2H), 7.56 (s, 1H), 7.44 (s, 1H), 7.35 (d, J=5.0 Hz, 1H), 7.29(d, J=8.3 Hz, 1H), 7.23 (d, J=3.1 Hz, 1H), 7.07-7.03 (m, 1H), 6.79 (d,J=8.4 Hz, 1H), 5.22 (s, 2H), 3.56 (s, 4H), 3.08 (d, J=22.9 Hz, 6H), 2.31(d, J=12.9 Hz, 2H), 1.56 (t, J=10.1 Hz, 2H), 1.42 (s, 3H). LCMS: m/z=522(M+H)⁺

Example 9: Synthesis of Compound 009

Step 1: A mixture of compound 1 (1.86 g, 10 mmol), compound Boc-amine(3.0 g, 15 mmol), and NEt3 (3.0 g, 30 mmol) in 1,4-dioxane (20 ml) wasstirred at 95° C. overnight. The mixture was concentrated, and followingthe addition of EA (60 ml) and of aqueous citric acid (60 ml), themixture was stirred for 30 min. The organic layer was separated, driedand concentrated to yield compound 2 (3.4 g, 97%) as a light yellowsolid.

Step 2: A mixture of compound 2 (3.5 g, 10 mmol) and NaOH (2M, 15 ml) inEtOH (15 ml) and THE (15 ml) was stirred at 60° C. for 2 h. The mixturewas concentrated, and following the addition of aqueous citric acid toadjust to pH<7, the mixture was stirred for 30 min, and filtered toafford compound 3 (2.8 g, 90%) as a light yellow solid.

Step 3: A mixture of compound 3 (3.2 g, 10 mmol), compound Boc-amine(2.9 g, 10 mmol), HOAT (2.0 g, 15 mmol), and EDCI (3.8 g, 20 mmol) inDMF (25 ml) was stirred at 60° C. overnight. To the mixture was added EA(100 ml) and aqueous saturated NaCl (100 ml), and the mixture wasstirred for 30 min. The organic layer was separated, washed by aqueoussaturated NaCl (50 ml*2), dried and concentrated to yield a residue,which was washed by CH3CN (10-20 ml) to afford compound 4 (2.9 g, 50%)as a gray solid.

Step 3: To a solution of compound 4 (2.9 g, 5 mmol) in DCM (30 ml) wasadded TFA (5 ml). The mixture was stirred at room temperature for 2 h.The mixture was concentrated to afford compound 5 (2.9 g, 100%) withoutany further purification.

Step 4: To a solution of compound 5 (197 mg, 0.5 mmol) and NEt3 (250 mg,2.5 mmol) in DCM (5 ml) was added piperidine-1-carbonyl chloride (96 mg,0.65 mmol) at 0° C. LCMS was monitored until reaction completion. To themixture was added NH3H2O (0.5 ml), the mixture was stirred for 30 minand was concentrated to get a residue, which was purified by silica gelcolumn to afford Compound 009 (114 mg, 45%) as light yellow solid. ¹HNMR (500 MHz, DMSO) δ 9.69 (s, 1H), 8.86 (s, 2H), 7.89 (d, J=8.0 Hz,1H), 7.49 (s, 1H), 7.40 (d, J=5.1 Hz, 1H), 7.36 (d, J=8.2 Hz, 1H), 7.30(d, J=3.3 Hz, 1H), 7.07 (dd, J=5.0, 3.7 Hz, 1H), 6.90 (d, J=8.2 Hz, 1H),3.99 (s, 1H), 3.56 (d, J=11.5 Hz, 2H), 3.10 (s, 4H), 2.84 (t, J=11.6 Hz,2H), 1.85 (d, J=10.7 Hz, 2H), 1.52 (s, 2H), 1.46 (d, J=8.3 Hz, 6H).LCMS: m/z=506 (M+H)⁺.

Example 10: Synthesis of Compound 010

Steps 1-7: Refer to steps 1-7 of Example 7 to obtain compound 8.

Step 8: To a solution of compound 8 (100 mg, 0.22 mmol) and Et3N (44 mg,0.45 mmol) in THE (5 ml) was added piperazine-1-carbonyl chloride (60mg, 0.24 mmol). The mixture was stirred at room temperature for 2 hours.After completion, the mixture was concentrated to afford the crudecompound 9 as an oil (110 mg, crude).

Step 9: To a solution of the compound 9 (100 mg) in DCM (5 ml) was addedTFA (1 ml) at room temperature for 40 mins. After completion, themixture was concentrated and purification on prep-HPLC to affordCompound 010 (35 mg, 30%, 2 steps) as a yellow solid. ¹H NMR (500 MHz,DMSO) δ 9.70 (s, 1H), 8.86 (s, 2H), 8.79 (s, 2H), 7.63 (s, 1H), 7.49 (d,J=1.8 Hz, 1H), 7.40 (d, J=5.0 Hz, 1H), 7.36 (dd, J=8.3, 1.9 Hz, 1H),7.29 (d, J=3.2 Hz, 1H), 7.10-7.04 (m, 1H), 6.90 (d, J=8.3 Hz, 1H), 3.37(s, 1H), 3.29 (s, 4H), 3.09 (s, 6H), 2.32 (d, J=13.6 Hz, 2H), 1.56 (t,J=10.2 Hz, 2H), 1.43 (s, 3H). LCMS: m/z=521 (M+H)⁺.

Example 11: Synthesis of Compound 011

Steps 1-7: Refer to steps 1-7 of Example 7 to obtain compound 8.

Step 8: To a solution of compound 8 (100 mg, 0.22 mmol) and Et3N (45 mg,0.45 mmol) in THE (5 ml) was added 4-methylpiperazine-1-carbonylchloride (40 mg, 0.24 mmol). The mixture was stirred at room temperaturefor 2 h. The mixture was filtered through silica gel and washed with EA.Concentration and purification by Prep-HPLC yielded Compound 011 (42 mg,36%). ¹H NMR (500 MHz, DMSO) δ 9.88 (s, 1H), 9.72 (s, 1H), 8.86 (s, 2H),7.64 (s, 1H), 7.49 (d, J=1.8 Hz, 1H), 7.41 (d, J=5.0 Hz, 1H), 7.37 (dd,J=8.3, 2.0 Hz, 1H), 7.30 (d, J=2.9 Hz, 1H), 7.08 (dd, J=4.9, 3.7 Hz,1H), 6.92 (d, J=8.4 Hz, 1H), 3.63 (d, J=11.2 Hz, 2H), 3.38 (d, J=8.2 Hz,3H), 3.15-2.97 (m, 6H), 2.81 (s, 3H), 2.33 (d, J=13.5 Hz, 2H), 1.57 (t,J=10.1 Hz, 2H), 1.43 (s, 3H). LCMS: m/z=535 (M+H)⁺.

Example 12: Synthesis of Compound 012

Step 1: To a solution of compound 6 (95 mg, 0.79 mmol) and Et3N (159 mg,1.6 mmol) in THE (5 ml) was added bis(trichloromethyl)carbonate (119 mg,0.4 mmol). The mixture was stirred at room temperature for 2 hours andwas used directly in the next step (step 2).

Step 2: Refer to steps 1-7 of Example 7 to obtain compound 8. A solutionof compound 8 (162 mg, 0.4 mmol) and Et3N (80 mg, 0.8 mmol) in THE (5ml) was added to a solution of the reaction mixture of step 1 containingcompound 7. The reaction was stirred at room temperature for 2 hours.Then the mixture was concentrated and purified by Pre-HPLC to yieldCompound 012 (35 mg, 16%). ¹H NMR (500 MHz, DMSO) δ 9.73 (s, 1H), 8.86(s, 2H), 7.62 (s, 1H), 7.50 (s, 1H), 7.42 (d, J=5.0 Hz, 1H), 7.38 (d,J=8.4 Hz, 1H), 7.31 (d, J=3.1 Hz, 1H), 7.08 (dd, J=5.0, 3.7 Hz, 1H),6.93 (d, J=8.1 Hz, 1H), 3.34 (s, 1H), 3.31 (s, 1H), 3.23 (d, J=5.6 Hz,4H), 3.08 (t, J=10.8 Hz, 2H), 2.31 (d, J=13.7 Hz, 2H), 1.94 (d, J=13.9Hz, 4H), 1.57 (t, J=10.0 Hz, 2H), 1.43 (s, 3H). LCMS: m/z=556 (M+H)⁺.

Example 13: Synthesis of Compound 013

Step 1: A mixture of methyl 4-iodobenzoate (2.6 g, 10 mmol), compound 4(6.4 g, 30 mmol), Pd2(dba)3 (915 mg, 1 mmol), Ruphos (467 mg, 1 mmol)and Cs2CO3 (9.75 g, 30 mmol, 3 eq) in tol (30 ml) was stirred at 95° C.under nitrogen atmosphere overnight. The mixture was cooled, and EA wasadded (100 ml). The mixture was filtered and was concentrated to get aresidue, which was purified by silica gel column to afford compound 5(2.2 g, 60%) as light yellow solid.

Step 2: To a solution of the compound 5 (500 mg, 1.4 mmol) in EtOH (15ml) and THE (15 ml) was added aqueous NaOH (2M, 15 ml), and the mixturewas stirred at 60° C. for 2 h. The mixture was concentrated to yield aresidue, and water (100 ml) was added and then aqueous citric acid wasadded to adjust to pH<7 at 0° C., then filtration afforded compound 6(369 mg, 80%) as a white solid.

Step 3: A mixture of compound 6 (150 mg, 0.45 mmol), compound Boc-amine(130 mg, 0.45 mmol), HOAT (103 mg, 0.76 mmol), EDCI (145 mg, 0.76 mmol)and NEt3 (154 mg, 1.52 mmol) in DMF (5 ml) was stirred at 60° C.overnight. To the mixture was added EA (100 ml) and aqueous saturatedNaCl (100 ml), and the mixture was stirred for 30 min. The organic layerwas separated, washed with aqueous saturated NaCl (50 ml*2), dried andconcentrated to get a residue, which was purified by Prep-TLC to affordcompound 7 (123 mg, 45%) as a yellow solid.

Step 4: To a solution of compound 7 (120 mg, 0.20 mmol) in DCM (5 ml)was added TFA (3 ml) at room temperature for 2 h. The mixture wasconcentrated to afford compound 8 (80 mg, 100%), which was used in thenext step without further purification.

Step 5: To a solution of the compound 8 (80 mg, 0.20 mmol) and Et3N (106mg, 1.05 mmol) in THE (5 ml) was added compound pyrrolidine-1-carbonylchloride (74 mg, 0.30 mmol) at 0° C. and stirred for 2 h. To the mixturewas added EA (50 ml) and aqueous saturated NaCl (50 ml), and the mixturewas stirred for 30 min. The organic layer was separated, washed byaqueous saturated NaCl (50 ml*2), dried and concentrated to afford aresidue which was purified by Prep-TLC to get Compound 013 (99 mg, 80%)as a yellow solid. ¹H NMR (500 MHz, DMSO) δ 9.35 (s, 1H), 7.75 (d, J=8.7Hz, 2H), 7.45 (d, J=2.0 Hz, 1H), 7.36 (d, J=5.1 Hz, 1H), 7.27 (dd,J=8.3, 2.1 Hz, 1H), 7.24 (d, J=3.5 Hz, 1H), 7.05 (dd, J=5.0, 3.6 Hz,1H), 6.79 (dd, J=8.5, 3.5 Hz, 3H), 5.89 (s, 1H), 5.07 (d, J=12.4 Hz,2H), 3.31 (d, J=7.8 Hz, 1H), 3.25 (t, J=6.4 Hz, 5H), 3.10 (t, J=10.5 Hz,2H), 1.97 (d, J=13.9 Hz, 2H), 1.74 (s, 4H), 1.59 (t, J=9.9 Hz, 2H), 1.37(s, 3H). LCMS: m/z=504 (M+H)⁺.

Example 14: Synthesis of Compound 014

Step 1: A solution of the 4-hydroxypiperidine (compound 1, 0.8 g, 7.9mmol), pyridine (1.3 g, 16 mmol) and tert-Butyldimethylsilyl chloride(1.4 g, 9.5 mmol) in CH2Cl2 (25 ml) was formed at 0° C. The mixture wasstirred for 3 h. After completion, the mixture was concentrated and waspurified by silica gel column with EA:PE=1:5 to afford compound 2 (1.5g, 88%) as a light yellow solid.

Step 2: To a solution of compound 2 (95 mg, 0.44 mmol) and Et3N (89 mg,0.9 mmol) in THE (5 ml) was added triphosgene (74 mg, 0.25 mmol). Themixture was stirred at room temperature for 2 hours and was useddirectly in the next step.

Step 3: To a solution of the compound 4 (162 mg, 0.4 mmol) and Et3N (80mg, 0.8 mmol) in THE (5 ml) was added the above solution from step 2containing compound 3. The mixture was stirred at room temperature for 2hours and was used in the next step without any further purification.

Step 4: To the solution of the compound 5 (crude mixture) in THE (5 ml)was added TBAF (5 drops) at 0° C. The mixture was stirred at roomtemperature for 2 hours, then concentrated and purified by Prep-HPLC toafford Compound 014 (29 mg, 15%, 3 steps). ¹H NMR (400 MHz, DMSO) δ 9.67(s, 1H), 8.86 (s, 2H), 7.56 (s, 1H), 7.48 (s, 1H), 7.39 (d, J=4.9 Hz,1H), 7.37-7.31 (m, 1H), 7.28 (d, J=3.3 Hz, 1H), 7.10-7.03 (m, 1H), 6.88(d, J=8.3 Hz, 1H), 3.59 (s, 3H), 3.42 (s, 1H), 3.25 (d, J=13.6 Hz, 2H),3.03 (t, J=11.0 Hz, 2H), 2.83 (t, J=10.6 Hz, 2H), 2.29 (d, J=13.5 Hz,2H), 1.70 (d, J=9.9 Hz, 2H), 1.57 (t, J=10.3 Hz, 2H), 1.44 (d, J=10.4Hz, 3H), 1.34-1.27 (m, 2H). LCMS: m/z=536 (M+H)⁺.

Example 15: Synthesis of Compound 015

Steps 1-4: Refer to steps 1-4 of Example 13 to obtain compound 8.

Step 5: To a solution of the compound 8 (80 mg, 0.20 mmol) and Et3N (106mg, 1.05 mmol) in THE (5 ml) was added tert-butyl4-(chlorocarbonyl)piperazine-1-carboxylate (74 mg, 0.30 mmol) at 0° C.,and the mixture was stirred for 2 h. To the mixture was added EA (20 ml)and aqueous saturated NaCl (20 ml), and the mixture was stirred for 30min. The organic layer was separated, washed by aqueous saturated NaCl(50 ml*2), dried and concentrated to yield a residue, which was purifiedby Prep-TLC to afford compound 9 (99 mg, 80%) as a yellow solid.

Step 6: To a solution of compound 9 (90 mg, 0.15 mmol) in DCM (5 ml) wasadded TFA (3 ml) slowly. The mixture was stirred at room temperature for2 h. Then the mixture was concentrated to get a residue, which waspurified by Prep-HPLC to yield Compound 015 (45 mg, 60%) as a whitesolid. ¹H NMR (400 MHz, DMSO) δ 9.58 (s, 1H), 8.78 (s, 2H), 7.78 (t,J=9.6 Hz, 2H), 7.52 (d, J=2.1 Hz, 1H), 7.42 (d, J=5.1 Hz, 1H), 7.36 (dd,J=8.3, 2.1 Hz, 1H), 7.31 (d, J=3.5 Hz, 1H), 7.08 (dd, J=5.0, 3.6 Hz,1H), 6.96 (d, J=8.3 Hz, 1H), 6.84 (d, J=8.4 Hz, 2H), 5.97 (s, 6H), 3.18(t, J=10.6 Hz, 2H), 3.10 (s, 4H), 2.02-1.93 (m, 2H), 1.60 (t, J=9.7 Hz,2H), 1.37 (s, 3H). LCMS: m/z=519 (M+H)⁺.

Example 16: Synthesis of Compound 016

Steps 1-4: Refer to steps 1-4 of Example 13 to obtain compound 8.

Step 5: To a solution of the compound 8 (80 mg, 0.20 mmol) and Et3N (106mg, 1.05 mmol) in THE (5 ml) was added 4-methylpiperazine-1-carbonylchloride (49 mg, 0.30 mmol) at 0° C., and the mixture was stirred for 2h. The mixture was purified by Prep-TLC to afford Compound 016 (49 mg,46%) as a yellow solid. ¹H NMR (400 MHz, DMSO) δ 9.79 (s, 1H), 9.58 (s,1H), 7.79 (d, J=8.7 Hz, 2H), 7.51 (d, J=2.1 Hz, 1H), 7.42 (d, J=4.2 Hz,1H), 7.36 (dd, J=8.3, 2.1 Hz, 1H), 7.31 (d, J=3.5 Hz, 1H), 7.08 (dd,J=5.0, 3.6 Hz, 1H), 6.95 (d, J=8.3 Hz, 1H), 6.84 (d, J=8.3 Hz, 2H), 3.64(d, J=11.6 Hz, 2H), 3.43-3.28 (m, 4H), 3.18 (t, J=10.3 Hz, 2H),3.10-2.96 (m, 4H), 2.81 (s, 3H), 1.99 (d, J=13.4 Hz, 2H), 1.60 (t, J=9.7Hz, 2H), 1.37 (s, 3H). LCMS: m/z=533 (M+H)⁺.

Example 17: Synthesis of Compound 017

Step 1: To a solution of lithium bis(trimethylsilyl)amide (1.0 M in THF,240 ml, 240 mmol) in a round-bottomed flask was added compound 1 (25 g,120 mmol) slowly at −76° C. under nitrogen atmosphere. After the mixturewas stirred for 4 hours at −76° C., iodoethane (17 ml, 240 mmol) wasadded dropwise into the system. The reaction mixture was stirred forfurther 30 minutes and then warmed to room temperature and stirredovernight. The residue was quenched with 150 ml saturated aqueous NH4Cl,diluted with water and extracted with EtOAc. The organic layers werewashed with water and brine then dried over sodium sulfate, filtered andconcentrated to afford target compound 2 (20 g, 70%) as a light yellowsolid.

Step 2: To a solution of the compound 2 (20 g, 84 mmol) was added K2CO3(23 g, 168 mmol) in DMSO (120 ml). H₂O₂ (100 ml) was added into thesystem at 60° C. very slowly and the reaction was stirred overnight at60° C. Cold water was added, and the mixture was extracted with EA(3*100 ml). The organic layers were washed with water and brine thendried over sodium sulfate, filtered and concentrated to get targetcompound 3 (21 g, 99%) as a white solid.

Step 3: To a solution of the compound 3 (20.5 g, 80 mmol) in CH3CN (200ml) and 5N KOH (100 ml) was added1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione (11.1 g, 40 mmol). Themixture was stirred overnight at room temperature. After completion, themixture was concentrated to remove CH3CN. Adjustment of the pH of thewater phase to 5 was with 2N HCl in ice bath, the mixture was extractedwith EA and separated. Then adjustment of the pH of water phase to 10.The precipitate was collected to afford the compound 4 (10 g, 55%) as awhite solid.

Step 4: A solution of the compound 4 (1.0 g, 4.4 mmol), ethyl4-bromobenzoate (943 mg, 4.4 mmol), Pd2(dba)3 (202 mg, 0.22 mmol),Ruphos (103 mg, 0.22 mmol) and Cs2CO3 (2.9 g, 8.8 mmol) in toluene (25ml) was stirred at 100° C. overnight. The mixture was concentrated andpurified by silica gel column with EA:PE=1:5 to afford compound 5 (1.0g, 59%) as a light yellow solid.

Step 5: A solution of the compound 5 (233 mg, 0.65 mmol) and 2N NaOH (10ml, 20 mmol) in THE (10 ml) and EtOH (10 ml) was stirred at 55° C. for 2hours. Concentration and adjustment of the pH of the water phase to 5-6,was followed by extraction with EA (3*15 ml). The organic layers werewashed with water and brine then dried over sodium sulfate, filtered andconcentrated to afford compound 6 (200 mg, 93%) as a white solid.

Step 6: A mixture of the compound 6 (200 mg, 0.6 mmol), tert-butyl2-amino-4-(thiophen-2-yl)phenylcarbamate (173 mg, 0.6 mmol), EDCI (154mg, 1.2 mmol) and DMAP (145 mg, 1.2 mmol) in DMF (10 ml) was stirred at55° C. overnight. The mixture was diluted with water and extracted withEA. The organic layers were washed with water and brine then dried oversodium sulfate, filtered and concentrated. Then purification by silicagel column with EA:PE=1:2 yielded compound 7 (200 mg, 55%) as a purplesolid.

Step 7: To a solution of compound 7 (200 mg, 0.32 mmol) in 1,4-dioxane(10 ml) was added HCl/1,4-dioxane (5 ml, 20 mmol), and the mixture wasstirred at room temperature overnight. Concentration and washing with PEafforded compound 8 (175 mg, 100%) as a gray solid.

Step 8: To the solution compound 8 (95 mg, 0.21 mmol) and Et3N (106 mg,1.05 mmol) in THE (5 ml) was added morpholine-4-carbonyl chloride (50mg, 0.3 mmol), and the mixture was stirred at room temperature for 2hours. The mixture was filtered through silica gel and was washed withEA. Concentration and purification by Prep-HPLC afforded Compound 017(10 mg, 9%). ¹H NMR (400 MHz, DMSO) δ 9.56 (s, 1H), 7.76 (d, J=8.7 Hz,2H), 7.52 (s, 1H), 7.42 (d, J=4.8 Hz, 1H), 7.36 (d, J=8.2 Hz, 1H), 7.31(d, J=3.0 Hz, 1H), 7.11-7.04 (m, 1H), 6.95 (d, J=8.2 Hz, 1H), 6.81 (d,J=8.5 Hz, 2H), 5.86 (s, 1H), 3.55 (s, 8H), 3.11 (s, 4H), 2.02 (d, J=13.3Hz, 2H), 1.76 (d, J=6.6 Hz, 2H), 1.50 (t, J=10.9 Hz, 2H), 0.75 (t, J=7.3Hz, 3H). LCMS: m/z=534 (M+H)⁺.

Example 18: Synthesis of Compound 018

Step 1: To a solution of compound 1 (1.2 g, 5 mmol) in dioxane (20 ml)was added DIPEA (1.3 g, 10 mmol) and ethyl2-chloropyrimidine-5-carboxylate (5.5 mmol, 1.0 g). The mixture wasstirred at 100° C. overnight. After completion, concentration and directpurification by prep-TLC with PE:EA=2:1 afforded compound 2 (1.37 g,70%) as a white solid.

Step 2: To a solution of compound 2 (220 mg, 0.56 mmol) in EtOH (5 ml)and THE (5 ml) was added aqueous NaOH (2M, 2 ml). The reaction wasstirred at 60° C. for 2 h. The mixture was concentrated to get aresidue. To the residue was added water (10 ml), and aqueous citric acidwas added to adjust to pH<7 at 0° C., and the mixture was filtered toafford compound 3 (200 mg, crude) as a white solid.

Step 3: A mixture of the compound 3 (200 mg, crude), compound tert-butyl2-amino-4-(thiophen-2-yl)phenylcarbamate (160 mg, 0.55 mmol), EDCI (154mg, 1.2 mmol) and DMAP (145 mg, 1.2 mmol) in DMF (10 ml) was stirred at67° C. overnight. The mixture was diluted with water and extracted withEA to afford compound 4 (200 mg, 55%) as a yellow oil.

Step 4: To the solution of compound 4 (200 mg, 0.32 mmol) in DCM (10 ml)was added TFA (2 ml), and the mixture was stirred at room temperaturefor 30 min. Concentration and washing with PE afforded compound 5 (250mg, crude) as a brown oil.

Step 5: To the solution of compound 5 (250 mg, crude) and Et3N (106 mg,1.05 mmol) in THE (5 ml) was added pyrrolidine-1-carbonyl chloride (70mg, 0.5 mmol). The mixture was stirred at room temperature for 2 hours.The mixture was filtered through silica gel and washed with EA.Concentration and purification by Prep-HPLC afforded Compound 018 (61mg, 60%). ¹H NMR (400 MHz, DMSO) δ 9.72 (s, 1H), 8.91 (s, 2H), 7.53 (s,1H), 7.42 (d, J=4.8 Hz, 1H), 7.39 (d, J=8.3 Hz, 1H), 7.32 (d, J=3.2 Hz,1H), 7.12-7.04 (m, 1H), 6.95 (d, J=8.2 Hz, 1H), 6.85-6.13 (m, 1H), 3.70(dd, J=13.5, 7.5 Hz, 4H), 3.29 (s, 4H), 3.07 (t, J=10.5 Hz, 2H), 2.81(t, J=12.8 Hz, 2H), 2.11 (dd, J=14.5, 7.9 Hz, 2H), 1.93-1.84 (m, 2H),1.76 (s, 4H), 1.32 (d, J=12.1 Hz, 2H). LCMS: m/z=532 (M+H)⁺.

Example 19: Synthesis of Compound 019

Step 1: A mixture of methyl 4-bromobenzoate (2.1 g, 10 mmol, 1 eq),compound 1 (6.0 g, 30 mmol, 3 eq), Pd2(dba)3 (915 mg, 1 mmol, 0.1 eq),Xantphos (478 mg, 1 mmol, 0.1 eq) and Cs2CO3 (9.75 g, 30 mmol, 3 eq) intoluene (30 ml) was stirred at 95° C. under nitrogen atmosphereovernight. The mixture was cooled, and addition of EA (100 ml),filtration and concentration yielded a residue, which was purified bysilica gel column to afford compound 2 (1.97 g, 59%) as a light yellowsolid.

Step 2: To a solution of compound 2 (3.34 g, 10 mmol) in EtOH (15 ml)and TH (15 ml) was added aqueous NaOH (2M, 15 ml) and stirred at 60° C.for 5 h. The mixture was concentrated to get a residue. The mixture wasdiluted with water (100 ml), and addition of aqueous citric acid to pH<7at 0° C. and filtration afforded compound 3 (3.07 g, 96%) as a whitesolid.

Step 3: A mixture of compound 3 (3.2 g, 10 mmol, 1 eq), Boc-amine (2.6g, 9 mmol, 0.9 eq), EDCI (5.7 g, 30 mmol, 3 eq) in Py (15 ml) wasstirred at 25° C. overnight. To the mixture was added EA (100 ml) andaqueous citric acid (50 ml), and the mixture was stirred for 30 mins.The organic layer was separated, dried and concentrated to yield aresidue, which was purified by silica gel column to afford compound 4(3.7 g, 70%) as a light yellow solid.

Step 4: To a solution of compound 4 (2.96 g, 5 mmol) in DCM (20 ml) wasadded TFA (20 ml), and the mixture was stirred at room temperature for 1h. The mixture was concentrated to yield a residue. To the mixture wasadded water (100 ml) and NaOH (2M solution) to adjust to pH>7, and themixture was filtered to yield compound 5 (1.86 g, 95%) as a light yellowsolid.

Step 5: To the solution of compound 5 (100 mg, 0.25 mmol) and Et3N (50mg, 0.5 mmol) in THE (5 ml) was added tert-butyl4-(chlorocarbonyl)piperazine-1-carboxylate (68 mg, 0.275 mmol), and themixture was stirred at room temperature for 2 hours. After completion,the mixture was concentrated to afford the crude compound 6 (110 mg) forthe next step.

Step 6: To a solution of compound 6 (110 mg, crude) in DCM (5 ml) wasadded TFA (2 ml) slowly. The mixture was stirred at room temperature for2 h. Then the mixture was concentrated to get a residue, which waspurified by Prep-HPLC to yield Compound 019 (40 mg, 32%, 2 steps) as awhite solid. ¹H NMR (400 MHz, DMSO) δ 9.37 (s, 1H), 8.71 (s, 2H), 7.79(d, J=8.7 Hz, 2H), 7.45 (d, J=2.1 Hz, 1H), 7.36 (d, J=5.1 Hz, 1H),7.30-7.20 (m, 3H), 7.05 (dd, J=5.1, 3.6 Hz, 1H), 6.81 (d, J=8.3 Hz, 1H),6.66 (d, J=8.8 Hz, 2H), 3.63 (d, J=13.5 Hz, 2H), 3.48-3.42 (m, 1H), 3.30(s, 4H), 3.11 (s, 4H), 2.96 (t, J=11.9 Hz, 2H), 1.92 (d, J=9.8 Hz, 2H),1.34 (d, J=10.8 Hz, 2H), 1.05 (t, J=7.0 Hz, 1H). LCMS: m/z=505 (M+H)⁺.

Example 20: Synthesis of Compound 020

Steps 1-4: Refer to steps 1-4 of Example 19 to obtain compound.

Step 5: To the solution of compound 5 (100 mg, 0.25 mmol) and Et3N (50mg, 0.5 mmol) in THE (5 ml) was added tert-butyl4-(chlorocarbonyl)piperazine-1-carboxylate (41 mg, 0.275 mmol), and themixture was stirred at room temperature for 2 hours. Then the mixturewas concentrated to get a residue, which was purified by Prep-HPLC toafford Compound 020 (32 mg, 25%) as a white solid. ¹H NMR (400 MHz,DMSO) δ 9.37 (s, 1H), 7.78 (d, J=8.6 Hz, 2H), 7.45 (d, J=2.0 Hz, 1H),7.36 (d, J=4.4 Hz, 1H), 7.30-7.20 (m, 2H), 7.05 (dd, J=5.0, 3.7 Hz, 1H),6.80 (d, J=8.3 Hz, 1H), 6.65 (d, J=8.8 Hz, 2H), 6.20 (d, J=7.9 Hz, 1H),5.07 (s, 2H), 3.60-3.52 (m, 6H), 3.18-3.06 (m, 4H), 2.92 (t, J=11.6 Hz,2H), 2.08 (s, 1H), 1.91 (d, J=10.2 Hz, 2H), 1.33 (dd, J=20.7, 9.8 Hz,2H). LCMS: m/z=506 (M+H)⁺.

Example 21: Synthesis of Compound 021

Steps 1-4: Refer to steps 1-4 of Example 19 to obtain compound 5.

Step 5: To the solution of compound 5 (100 mg, 0.25 mmol) and Et3N (50mg, 0.5 mmol) in THE (5 ml) was added cyclohexanecarbonyl chloride (41mg, 0.275 mmol), and the mixture was stirred at room temperature for 2hours. Then the mixture was concentrated to get a residue, which waspurified by Prep-HPLC to afford Compound 021 (30 mg, 24%) as a whitesolid. ¹H NMR (400 MHz, DMSO) δ 9.61 (s, 1H), 7.81 (d, J=8.6 Hz, 2H),7.53 (d, J=1.8 Hz, 1H), 7.43 (d, J=4.8 Hz, 1H), 7.38 (d, J=8.3 Hz, 1H),7.33 (d, J=3.3 Hz, 1H), 7.13-7.05 (m, 1H), 6.98 (d, J=8.4 Hz, 1H), 6.68(d, J=8.7 Hz, 2H), 4.29 (d, J=11.3 Hz, 1H), 3.92 (d, J=12.5 Hz, 1H),3.66-3.57 (m, 1H), 3.27-3.13 (m, 1H), 2.85-2.73 (m, 1H), 2.67-2.56 (m,1H), 2.03-1.88 (m, 2H), 1.75-1.58 (m, 5H), 1.37-1.12 (m, 7H). LCMS:m/z=503 (M+H)⁺.

Example 22: Synthesis of Compound 022

Steps 1-4: Refer to steps 1-4 of Example 19 to obtain compound 5.

Step 5: To the solution of compound 5 (100 mg, 0.25 mmol) and Et3N (50mg, 0.5 mmol) in THE (5 ml) was added tetrahydro-2H-pyran-4-carbonylchloride (41 mg, 0.275 mmol), and the mixture was stirred at roomtemperature for 2 hours. Then the mixture was concentrated to get aresidue, which was purified by Prep-HPLC to afford Compound 022 (20 mg,16%) as a white solid. ¹H NMR (400 MHz, DMSO) δ 9.57 (s, 1H), 7.80 (d,J=8.6 Hz, 2H), 7.52 (s, 1H), 7.46-7.29 (m, 3H), 7.1-7.06 (m, 1H), 6.96(s, 1H), 6.68 (d, J=8.6 Hz, 2H), 4.29 (d, J=14.4 Hz, 1H), 3.97 (d,J=13.9 Hz, 1H), 3.85 (d, J=9.7 Hz, 2H), 3.71-3.55 (m, 1H), 3.39 (t,J=11.5 Hz, 2H), 3.22 (t, J=11.6 Hz, 1H), 2.98-2.87 (m, 1H), 2.81 (t,J=12.1 Hz, 1H), 2.08-1.86 (m, 2H), 1.68-1.43 (m, 4H), 1.39-1.13 (m, 2H).LCMS: m/z=505 (M+H)⁺.

Example 23: Synthesis of Compound 023

Steps 1-7: Refer to steps 1-7 of Example 17 to obtain compound 8.

Step 8: To a mixture of the compound 8 (175 mg, 0.4 mmol) and Et3N (106mg, 1.05 mmol) in THE (5 ml) was added tert-butyl 4-(chlorocarbonyl)piperazine-1-carboxylate (125 mg, 0.5 mmol). The mixture was stirred atroom temperature for 2 hours. Filtration through silica gel and washingwith EA yielded compound 9 (175 mg, 67%) as a yellow oil.

Step 9: To the solution of the compound 9 (175 mg, 0.28 mmol) in1,4-dioxane (10 ml) was added HCl/1,4-dioxane (5 ml, 20 mmol) at roomtemperature followed by stirring overnight. Concentration and washingwith PE to afforded target Compound 023 (92 mg, 63%) as a yellow solid.¹H NMR (400 MHz, DMSO) δ 10.21 (s, 1H), 9.30 (s, 3H), 7.91 (d, J=8.1 Hz,2H), 7.81 (s, 1H), 7.59 (d, J=5.9 Hz, 2H), 7.51 (d, J=3.3 Hz, 1H), 7.45(d, J=8.4 Hz, 1H), 7.19-7.12 (m, 1H), 6.90 (s, 2H), 3.48-3.41 (m, 1H),3.33 (s, 4H), 3.18-3.10 (m, 2H), 3.06 (s, 4H), 2.04 (d, J=13.0 Hz, 2H),1.77 (d, J=7.0 Hz, 2H), 1.59-1.48 (m, 2H), 1.08-1.02 (m, 2H), 0.78 (t,J=7.0 Hz, 3H). LCMS: m/z=533 (M+H)⁺.

Example 24: Synthesis of Compound 024

Step 1: A solution of the compound 2 (300 mg, 0.90 mmol) in TFA (5 ml)was stirred at room temperature overnight. Then concentration andwashing with PE yielded target compound 3 (200 mg, 95%) as a gray solid.

Step 2: A mixture of the compound 3 (180 mg, 0.77 mmol),1-hydroxycyclohexane-carboxylic acid (111 mg, 0.77 mmol), HATU (351 mg,0.92 mmol) and DIPEA (199 mg, 1.5 mmol) in DMF (10 ml) was formed. Thereaction was stirred at room temperature for 2 hours. The mixture wasdissolved in water and extracted with EA. The organic layers were washedwith water and brine then dried over sodium sulfate, filtered andconcentrated. Then purification by silica gel column with EA:PE=1:2yielded compound 4 (150 mg, 54%) as a purple solid.

Step 3: The solution of compound 4 (150 mg, 0.42 mmol) and 2N NaOH (10ml, 20 mmol) in THE (10 ml) and EtOH (10 ml) was stirred at 60° C. for 6hours. Then the mixture was concentrated, and the pH of the water phasewas adjusted to 5-6 followed by extraction with EA. The organic layerswere washed with water and brine then dried over sodium sulfate,filtered and concentrated to yield the target compound 5 (130 mg, 90%)as a white solid.

Step 4: A mixture of the compound 5 (70 mg, 0.2 mmol), tert-butyl2-amino-4-(thiophen-2-yl)phenylcarbamate (59.0 mg, 0.2 mmol), EDCI (39.0mg, 0.3 mmol), HOAT (41 mg, 0.3 mmol) and DIPEA (52 mg, 0.4 mmol) in DMF(10 ml) was formed. The mixture was stirred at 65° C. overnight. Thenthe mixture was dissolved in water and extracted with EA. The organiclayers were washed with water and brine then dried over sodium sulfate,filtered and concentrated. Then purification by silica gel column withEA:PE=1:2 yielded compound 6 (20 mg, 16%) as a purple solid.

Step 5: To the solution of the compound 6 (20 mg, 0.03 mmol) in DCM (10ml) was added HCl/1,4-dioxane (2 ml, 8.0 mmol) followed by stirring atroom temperature overnight. Concentration and washing with PE affordedthe target Compound 024 (12 mg, 71%) as a white solid. ¹H NMR (400 MHz,DMSO) δ 9.41 (s, 1H), 7.80 (d, J=8.5 Hz, 2H), 7.46 (s, 1H), 7.36 (d,J=4.9 Hz, 1H), 7.30-7.22 (m, 2H), 7.09-7.03 (m, 1H), 6.80 (d, J=8.4 Hz,1H), 6.66 (d, J=8.6 Hz, 2H), 6.26 (d, J=7.9 Hz, 1H), 5.24 (s, 1H), 5.09(s, 2H), 2.04-1.87 (m, 3H), 1.76-1.39 (m, 10H), 1.35-1.14 (m, 6H). LCMS:m/z=519 (M+H)⁺.

Example 25: Synthesis of Compound 025

Step 1: Refer to step 1 of Example 24 to obtain compound 3.

Step 2: A mixture of the compound 3 (400 mg, 1.7 mmol),1-hydroxycyclopentane-carboxylic acid (222 mg, 1.7 mmol), HATU (969 mg,2.6 mmol) and DIPEA (439 mg, 3.4 mmol) in DMF (10 ml) was formed. Themixture was stirred at room temperature for 2 hours. The mixture wasdissolved in water and extracted with EA. organic layers were washedwith water and brine then dried over sodium sulfate, filtered andconcentrated. Then purification by silica gel column with EA:PE=1:2yielded compound 4 (300 mg, 51%) as a purple solid.

Step 3: To a solution of compound 4 (300 mg, 0.87 mmol) in THE (10 ml)and EtOH (10 ml) was added 2N NaOH (10 ml, 20 mmol) at 60° C., and theresulting reaction mixture was stirred for 6 hours. Concentration andadjustment of the pH of the water phase to 5-6 was followed byextraction with EA (2*15 ml). The organic layers were washed with waterand brine then dried over sodium sulfate, filtered and concentrated toyield target compound 5 (250 mg, 87%) as a white solid.

Step 4: A mixture of the compound 5 (250 mg, 0.75 mmol), tert-butyl2-amino-4-(thiophen-2-yl)phenylcarbamate (218 mg, 0.75 mmol), EDCI(143.0 mg, 1.1 mmol), HOAT (147 mg, 1.1 mmol) and DIPEA (194 mg, 1.5mmol) in DMF (10 ml) was formed. The reaction was stirred at 65° C.overnight. Then the mixture was dissolved in water and extracted withEA. The organic layers were washed with water and brine then dried oversodium sulfate, filtered and concentrated. Then purification by silicagel column with EA:PE=1:2 yielded compound 6 (50 mg, 11%) as a purplesolid.

Step 5: To a solution of the compound 6 (50 mg, 0.08 mmol) in DCM wasadded HCl/1,4-dioxane (2 ml, 8.0 mmol). The mixture was stirred at roomtemperature overnight. Concentration and washing with PE afforded thetarget Compound 025 (13 mg, 32%) as a white solid. ¹H NMR (400 MHz,DMSO) δ 9.61 (s, 1H), 7.81 (d, J=8.6 Hz, 2H), 7.53 (s, 1H), 7.46-7.30(m, 3H), 7.11-7.07 (m, 1H), 6.98 (d, J=8.0 Hz, 1H), 6.68 (d, J=8.7 Hz,2H), 4.57-4.44 (m, 1H), 4.36-4.19 (m, 1H), 3.24-3.17 (m, 1H), 2.90-2.79(m, 1H), 2.39-2.25 (m, 1H), 2.08-1.91 (m, 4H), 1.74-1.63 (m, 4H),1.57-1.52 (m, 2H), 1.38-1.22 (m, 2H). LCMS: m/z=505 (M+H)⁺.

Example 26: Synthesis of Compound 026

Step 1: To a mixture of compound 1 (300 mg, 0.88 mmol) in THE (5 ml) andEtOH (5 ml) was added 2M NaOH (over 5 m), and the resulting reactionmixture was stirred at 60° C. for 5 h. The mixture was concentrated toafford a residue, which was purified by flash column to yield compound 2(180 mg, yield: 65%) as a yellow solid.

Step 2: A mixture of compound 2 (150 mg, 0.48 mmol), compound Boc-amine(139 mg, 0.48 mmol), HOAT (131 mg, 096 mmol), and EDCI (183 mg, 0.96mmol) in DMF (4 ml) was stirred at 60° C. overnight. To the mixture wasadded EA (30 ml) and aqueous saturated NaCl (100 ml), and the resultingreaction mixture was stirred for 30 min. The organic layer wasseparated, washed with aqueous saturated NaCl (50 ml*2), dried andconcentrated to afford a residue, which was purified by Prep-TLC toyield compound 3 (100 mg, 35%) as a yellow solid.

Step 3: To a solution of compound 3 (100 mg, 0.17 mmol) in DCM (3 ml)was added TFA (3 ml), and the resulting reaction mixture was stirred atroom temperature for 2 h. The mixture was concentrated to get a residue,which was purified by Prep-HPLC to afford Compound 026 (20 mg, 24%) as awhite solid. ¹H NMR (400 MHz, DMSO) δ 9.53 (s, 1H), 8.76 (d, J=86.5 Hz,2H), 8.41 (d, J=13.8 Hz, 1H), 7.43-7.38 (m, 3H), 7.37-7.31 (m, 3H), 7.29(dd, J=8.3, 2.2 Hz, 1H), 7.22 (dd, J=7.7, 4.2 Hz, 2H), 7.04 (dd, J=5.1,3.6 Hz, 1H), 6.79 (d, J=8.4 Hz, 1H), 3.46 (d, J=11.2 Hz, 2H), 3.34-3.20(m, 2H), 2.96-2.90 (m, 2H), 2.86 (d, J=4.5 Hz, 3H), 2.26-2.08 (m, 2H).LCMS: m/z=485 (M+H)⁺.

Example 27: Synthesis of Compound 027

Step 1: A mixture of compound 1 (3.0 g, 15 mmol), bromobenzene (7.0 g,45 mmol), Pd2(dba)3 (1.4 g, 1.5 mmol), Ruphos (700 mg, 1.5 mmol) andCs2CO3 (14.6 g, 45 mmol) in toluene (100 ml) was stirred at 95° C. undera nitrogen atmosphere overnight. The mixture was filtered andconcentrated followed by washing with PE (30 ml) to afford compound 2(3.5 g, yield: 85%) as a light yellow solid.

Step 2: To a solution of compound 2 (3.5 g, 12.6 mmol) in 1,4-dioxane(50 ml) was added HCl in 1,4-dioxane (9.45 ml, 37.8 mmol) at roomtemperature, and the mixture was stirred overnight. The mixture wasfiltered to afford compound 3 (2.0 g, 90%) as a white solid.

Step 3: A mixture of compound 3 (500 mg, 2.8 mmol), ethyl2-chloropyrimidine-5-carboxylate (352 mg, 1.9 mmol), and NEt3 (576 mg,5.7 mmol) in 1,4-dioxane (15 ml) was stirred at 60° C. overnight. To themixture was added EA (100 ml) and aqueous saturated citric acid (30 ml),and the resulting reaction mixture was stirred for 30 min. The organiclayer was separated, washed by aqueous saturated NaCl (50 ml*2), driedand concentrated, and washed by PE (30 ml) to yield compound 4 (500 mg,81%) as a yellow solid.

Step 4: To a solution of compound 4 (500 mg, 1.5 mmol) in EtOH (15 ml)and THE (15 ml) was added aqueous NaOH (2M, 15 ml), and the resultingreaction mixture was stirred at 60° C. for 5 h. To the mixture was addedaqueous saturated citric acid to adjust to pH<7 followed by filtrationto yield compound 5 (400 mg, 87%) as a white solid.

Step 5: A mixture of compound 5 (150 mg, 0.5 mmol), compound Boc-amine(145 mg, 0.5 mmol), HOAT (136 mg, 1 mmol), EDCI (191 mg, 1 mmol) andNEt3 (202 mg, 2 mmol) in DMF (5 ml) was stirred at 60° C. overnight. Tothe mixture was added EA (100 ml) and aqueous saturated NaCl (100 ml),and the resulting reaction mixture was stirred for 30 min. The organiclayer was separated, washed by aqueous saturated NaCl (50 ml*2), driedand concentrated, and washed by CH3CN (10-20 mL) to afford compound 6(140 mg, 49%) as a yellow solid.

Step 6: To a solution of compound 6 (140 mg, 0.25 mmol) in DCM (5 ml)was added TFA (3 ml) at room temperature, and the resulting reactionmixture was stirred for 2 h. The mixture was concentrated and purifiedby Prep-HPLC to afford Compound 027 (110 mg, 95%) as a white solid. ¹HNMR (500 MHz, DMSO) δ 9.52 (s, 1H), 8.86 (s, 2H), 7.87 (d, J=7.8 Hz,1H), 7.45 (s, 1H), 7.35 (d, J=4.9 Hz, 1H), 7.29 (d, J=8.3 Hz, 1H),7.25-7.18 (m, 3H), 7.07-7.02 (m, 1H), 6.96 (d, J=8.2 Hz, 2H), 6.83-6.73(m, 2H), 5.20 (s, 2H), 3.72 (d, J=12.2 Hz, 2H), 2.82 (t, J=11.7 Hz, 2H),1.99 (s, 1H), 1.96 (d, J=10.5 Hz, 2H), 1.69-1.58 (m, 2H). LCMS: m/z=471(M+H)⁺.

Example 28: Synthesis of Compound 028

Steps 1-2: Refer to steps 1-2 of Example 27 to obtain compound 3.

Step 3: A mixture of compound 3 (300 mg, 1.7 mmol), 4-fluorobenzonitrile(181 mg, 1.5 mmol), and K2CO3 (414 mg, 3 mmol) in DMSO (10 ml) wasstirred at 100° C. overnight. To the mixture was added EA (100 ml) andaqueous saturated citric acid (30 ml), and the resulting reactionmixture was stirred for 30 min. The organic layer was separated, washedby aqueous saturated NaCl (50 ml*2), dried and concentrated, andpurified by silica gel column to yield compound 4 (300 mg, 72%) as ayellow solid.

Step 4: A solution of compound 4 (300 mg, 1.1 mmol) in 6M HCl (20 ml)was stirred at 80° C. for 3 days. The mixture was filtered to affordcompound 5 (250 mg, 78%) as a white solid.

Step 5: A mixture of compound 5 (150 mg, 0.5 mmol), compound Boc-amine(145 mg, 0.5 mmol), HOAT (136 mg, 1 mmol), EDCI (191 mg, 1 mmol) andNEt3 (202 mg, 2 mmol) in DMF (5 ml) was stirred at 60° C. overnight. Tothe mixture was added EA (80 ml) and aqueous saturated NaCl (80 ml), andthe resulting reaction mixture was stirred for 30 min. The organic layerwas separated, washed by aqueous saturated NaCl (30 ml*2), dried andconcentrated, and purified by Prep-TLC to afford compound 6 (50 mg, 18%)as a yellow solid.

Step 6: To a solution of compound 6 (50 mg, 0.09 mmol) in DCM (5 ml) wasadded TFA (3 ml) at room temperature, and the resulting reaction mixturewas stirred for 2 h. The mixture was concentrated and purified byPrep-HPLC to afford Compound 028 (5 mg, 12%) as a white solid. ¹H NMR(500 MHz, DMSO) δ 9.36 (s, 1H), 7.79 (d, J=8.6 Hz, 2H), 7.46 (d, J=1.9Hz, 1H), 7.35 (d, J=5.0 Hz, 1H), 7.27 (dd, J=8.3, 2.0 Hz, 1H), 7.25-7.18(m, 3H), 7.08-7.03 (m, 1H), 6.96 (d, J=8.2 Hz, 2H), 6.80 (d, J=8.3 Hz,1H), 6.75 (t, J=7.2 Hz, 1H), 6.67 (d, J=8.7 Hz, 2H), 6.22 (d, J=8.0 Hz,1H), 5.07 (s, 2H), 3.70 (d, J=12.8 Hz, 2H), 3.53 (s, 1H), 2.89 (t,J=11.0 Hz, 2H), 2.01 (d, J=10.8 Hz, 2H), 1.51 (dd, J=20.6, 10.5 Hz, 2H).LCMS: m/z=469 (M+H)⁺.

Example 29: Synthesis of Compound 029

Steps 1-2: Refer to steps 1-2 of Example 13 to obtain compound 6.

Step 3: A mixture of compound 6 (630 mg, 1.89 mmol), compound Boc-amine(484 mg, 1.7 mmol), HOAT (510 mg, 3.78 mmol), EDCI (721 mg, 3.78 mmol)DIPEA (487 mg, 3.78 mmol) and DMAP (461 mg, 3.78 mmol) in DMF (25 ml)was stirred at 67° C. for 3 days. To the mixture was added EA (100 ml)and aqueous saturated NaCl (100 ml), and the resulting reaction mixturewas stirred for 30 min. The organic layer was separated, washed byaqueous saturated NaCl (50 ml*2), dried and concentrated, and purifiedby silica gel column to afford compound 7 (473 mg, 46%) as a yellowsolid.

Step 4: To a solution of compound 7 (250 mg, 0.42 mmol) in DCM (10 ml)was added TFA (3 ml) at room temperature, and the resulting reactionmixture was stirred for 2 h. The mixture was concentrated to affordcompound 8 (300 mg, crude) and was used in the next step without furtherpurification.

Step 5: To a solution of the compound 8 (150 mg, crude) and Et3N (50 mg,0.5 mmol) in THF (5 ml) was added compound morpholine-4-carbonylchloride (37 mg, 0.25 mmol) at 0° C., and the resulting reaction mixturewas stirred for 2 h. To the mixture was added EA (10 ml) and aqueoussaturated NaCl (10 ml), and the resulting reaction mixture was stirredfor 30 min. The organic layer was separated, washed by aqueous saturatedNaCl (10 ml*2), dried and concentrated, and purified by Prep-HPLC toafford Compound 029 (44 mg, 35%) as a white solid. ¹H NMR (400 MHz,DMSO) δ 9.36 (s, 1H), 8.51 (dd, J=4.7, 1.5 Hz, 2H), 7.76 (d, J=8.8 Hz,2H), 7.66 (d, J=2.1 Hz, 1H), 7.57 (dd, J=4.7, 1.6 Hz, 2H), 7.47 (dd,J=8.4, 2.2 Hz, 1H), 6.88 (d, J=8.4 Hz, 1H), 6.79 (d, J=8.8 Hz, 2H), 5.87(s, 1H), 5.29 (s, 2H), 3.60-3.51 (m, 4H), 3.32-3.24 (m, 2H), 3.17-3.08(m, 6H), 1.98 (d, J=13.8 Hz, 2H), 1.64-1.55 (m, 2H), 1.36 (s, 3H). LCMS:m/z=515 (M+H)⁺.

Example 30: Synthesis of Compound 030

Steps 1-3: Refer to steps 1-3 of Example 29 to obtain compound 8.

Step 3: To a solution of the compound 8 (150 mg, crude) and Et3N (50 mg,0.5 mmol) in THF (5 ml) was added compound pyrrolidine-1-carbonylchloride (33 mg, 0.25 mmol) at 0° C., and the resulting reaction mixturewas stirred for 2 h. To the mixture was added EA (10 ml) and aqueoussaturated NaCl (10 ml), and the resulting reaction mixture was stirredfor 30 min. The organic layer was separated, washed by aqueous saturatedNaCl (10 ml*2), dried and concentrated, and purified by Prep-HPLC toafford Compound 030 (17 mg, 14%) as a white solid. ¹H NMR (400 MHz,DMSO) δ 9.34 (d, J=10.2 Hz, 1H), 8.51 (d, J=5.5 Hz, 2H), 7.76 (d, J=8.6Hz, 2H), 7.67 (d, J=1.8 Hz, 1H), 7.58 (d, J=5.9 Hz, 2H), 7.48 (dd,J=8.3, 1.9 Hz, 1H), 6.88 (d, J=8.4 Hz, 1H), 6.79 (d, J=8.6 Hz, 2H), 5.87(s, 1H), 5.31 (s, 2H), 3.31-3.20 (m, 6H), 3.10 (t, J=10.5 Hz, 2H), 1.98(d, J=13.5 Hz, 2H), 1.74 (s, 4H), 1.59 (t, J=10.1 Hz, 2H), 1.37 (s, 3H).LCMS: m/z=499 (M+H)⁺.

Example 31: Synthesis of Compound 031

Step 1: A mixture of compound 6 (270 mg, 0.8 mmol), compound Boc-amine(205 mg, 0.72 mmol), HOAT (216 mg, 1.6 mmol), EDCI (305 mg, 1.6 mmol)DIPEA (206 mg, 1.6 mmol) and DMAP (195 mg, 1.6 mmol) in DMF (25 ml) wasstirred at 67° C. for 3 days. To the mixture was added EA (60 ml) andaqueous saturated NaCl (60 ml), and the resulting reaction mixture wasstirred for 30 min. The organic layer was separated, washed by aqueoussaturated NaCl (20 ml*2), dried and concentrated, and purified by silicagel column to afford compound 7 (400 mg, 83%) as a yellow solid.

Step 2: To a solution of compound 7 (200 mg, 0.33 mmol) in DCM (10 ml)was added TFA (3 ml), and the resulting reaction mixture was stirred atroom temperature for 2 h. The mixture was concentrated to affordcompound 8 (250 mg, crude) and was used in the next step without furtherpurification.

Step 3: To a solution of the compound 8 (150 mg, crude) and Et3N (50 mg,0.5 mmol) in THF (5 ml) was added compound morpholine-4-carbonylchloride (37 mg, 0.25 mmol) at 0° C., and the resulting reaction mixturewas stirred for 2 h. To the mixture was added EA (10 ml) and aqueoussaturated NaCl (10 ml), and the resulting reaction mixture was stirredfor 30 min. The organic layer was separated, washed by aqueous saturatedNaCl (10 ml*2), dried and concentrated, and purified by Prep-HPLC toafford Compound 031 (55 mg, 60%) as a white solid. ¹H NMR (400 MHz,DMSO) δ 9.57 (s, 1H), 9.09 (s, 1H), 8.76-8.58 (m, 2H), 8.01-7.69 (m,4H), 7.56 (d, J=7.2 Hz, 1H), 7.05-6.87 (m, 3H), 3.56 (s, 4H), 3.17 (d,J=39.8 Hz, 8H), 1.96 (s, 2H), 1.61 (s, 2H), 1.37 (s, 3H). LCMS: m/z=515(M+H)⁺.

Example 32: Synthesis of Compound 032

Steps 1-2: Refer to steps 1-2 of Example 19 to obtain compound 3.

Step 3: A mixture of compound 3 (150 mg, 0.45 mmol), compound Boc-amine(130 mg, 0.45 mmol), HOAT (103 mg, 0.76 mmol), EDCI (145 mg, 0.76 mmol)and NEt3 (154 mg, 1.52 mmol) in CH3CN (5 ml) was stirred at 60° C.overnight. To the mixture was added EA (10 ml) and aqueous saturatedNaCl (10 ml), and the resulting reaction mixture was stirred for 30 min.The organic layer was separated, washed by aqueous saturated NaCl (10ml*2), dried and concentrated, and purified by silica gel column toafford compound 4 (90 mg, 30%) as a yellow solid.

Step 4: To a solution of compound 4 (90 mg, 0.15 mmol) in DCM (5 ml) wasadded TFA (3 ml) at room temperature, and the resulting reaction mixturewas stirred for 2 h. The mixture was concentrated to afford compound 5(58 mg, crude) and was used in the next step without furtherpurification.

Step 5: To a solution of the compound 5 (58 mg, 0.15 mmol) and Et3N (33mg, 0.33 mmol) in THE (5 ml) was added compound pyrrolidine-1-carbonylchloride (20 mg, 015 mmol) at 0° C., and the resulting reaction mixturewas stirred for 2 h. The mixture was purified by Prep-HPLC to affordCompound 032 (12 mg, 17%) as a white solid. ¹H NMR (400 MHz, DMSO) δ9.37 (s, 1H), 8.58 (d, J=5.1 Hz, 2H), 7.79 (d, J=8.8 Hz, 4H), 7.76 (d,J=2.1 Hz, 1H), 7.59 (dd, J=8.5, 2.2 Hz, 1H), 6.89 (d, J=8.5 Hz, 1H),6.66 (d, J=8.8 Hz, 2H), 6.22 (d, J=7.4 Hz, 1H), 3.63 (d, J=13.2 Hz, 2H),3.26 (t, J=6.4 Hz, 4H), 2.87 (t, J=11.3 Hz, 2H), 1.91 (d, J=10.0 Hz,2H), 1.75 (t, J=6.5 Hz, 4H), 1.34 (dd, J=20.2, 10.4 Hz, 2H). LCMS:m/z=485 (M+H)⁺.

Example 33: Synthesis of Compound 033

Step 1: A mixture of compound 1 (2.1 g, 9.6 mmol), ethyl2-chloropyrimidine-5-carboxylate (600 mg, 3.2 mmol) and NEt3 (970 mg,9.6 mmol) in 1,4-dioxane (20 ml) was stirred at 95° C. overnight. Themixture was concentrated followed by addition of EA (60 ml) and aqueouscitric acid (60 ml), and the resulting reaction mixture was stirred for30 min. The organic layer was separated, dried and concentrated toafford compound 2 (880 mg, yield: 75%) as a light yellow solid.

Step 2: To a solution of compound 2 (880 mg, 2.4 mmol) in DCM (10 ml)was added TFA (5 ml) at room temperature, and the resulting reactionmixture was stirred for 2 h. The mixture was concentrated to affordcompound 3 (630 mg, 99%) as a yellow solid.

Step 3: A mixture of compound 3 (630 mg, 2.4 mmol), 2-indoleacetic acid(531 mg, 3 mmol), HOAT (816 mg, 6 mmol), EDCI (1.1 g, 6 mmol) and NEt3(1.2 g, 12 mmol) in DMF (15 ml) was stirred at 60° C. overnight. To themixture was added EA (100 ml) and aqueous saturated NaCl (100 ml), andthe resulting reaction mixture was stirred for 30 min. The organic layerwas separated, washed by aqueous saturated NaCl (50 ml*2), dried andconcentrated to get a residue, which was purified by Prep-TLC to yieldcompound 4 (600 mg, 60%) as a yellow solid.

Step 4: To a solution of compound 4 (600 mg, 1.4 mmol) in EtOH (15 ml)and THE (15 ml) was added aqueous NaOH (2M, 15 ml), and the resultingreaction mixture was stirred at 60° C. for 5 h. The mixture wasconcentrated to yield a residue. To the mixture was added water (100 ml)and aqueous citric acid to adjust to pH<7 at 0° C., followed byfiltration to yield compound 5 (400 mg, 72%) as a white solid.

Step 5: A mixture of compound 5 (150 mg, 0.38 mmol), compound Boc-amine(108 mg, 0.38 mmol), HOAT (103 mg, 0.76 mmol), EDCI (145 mg, 0.76 mmol)and NEt3 (154 mg, 1.52 mmol) in DMF (5 ml) was stirred at 60° C.overnight. To the mixture was added EA (20 ml) and aqueous saturatedNaCl (20 mL), and the resulting reaction mixture was stirred for 30 min.The organic layer was separated, washed by aqueous saturated NaCl (20ml*2), dried and concentrated, and purified by Prep-TLC to affordcompound 6 (150 mg, 59%) as a yellow solid.

Step 6: To a solution of compound 6 (150 mg, 0.23 mmol) in DCM (5 ml)was added TFA (3 ml) at room temperature, and the resulting reactionmixture was stirred for 2 h. The mixture was concentrated and purifiedby Prep-HPLC to afford Compound 033 (28 mg, 22%) as a white solid. ¹HNMR (500 MHz, DMSO) δ 10.94 (s, 1H), 9.75 (s, 1H), 8.91 (s, 2H), 7.58(dd, J=15.8, 7.9 Hz, 5H), 7.41 (t, J=7.6 Hz, 4H), 7.36 (d, J=8.1 Hz,1H), 7.31-7.24 (m, 3H), 7.08 (t, J=7.5 Hz, 1H), 7.01-6.97 (m, 2H), 4.84(d, J=11.4 Hz, 2H), 4.58 (d, J=14.0 Hz, 2H), 4.14 (d, J=11.9 Hz, 1H),3.82 (q, J=15.1 Hz, 3H), 3.09 (t, J=12.1 Hz, 1H), 2.82 (s, 3H), 2.63 (t,J=12.0 Hz, 1H), 1.63-1.44 (m, 5H), 1.31 (d, J=8.2 Hz, 2H). LCMS: m/z=562(M+H)⁺.

Example 34: Synthesis of Compound 034

Step 1: Refer to step 1 of Example 3 to obtain compound 2.

Step 2: To a solution of compound 2 (5.25 g, 15 mmol) in DMSO (50 ml)was added K2CO3 (4.14 g, 30 mmol) and Cu (960 mg, 15 mmol). The mixturewas stirred under nitrogen atmosphere at 145° C. overnight. The mixturewas filtered, and the filtrate was collected followed by purification ona column to afford the compound 3 (2.4, 38%) as a white solid.

Step 3: To a solution of compound 3 (600 mg, 1.5 mmol) in 1,4-dioxane(15 ml) was added 4M HCl in 1,4-dioxane (10 ml) at room temperatureovernight. The mixture was filtered to get compound 4 (437 mg, 95%) as ayellow solid.

Step 4: A mixture of compound 4 (437 mg, 1.75 mmol), EDCI (543 mg, 3.5mmol), HOAT (476 mg, 3.5 mmol), DIPEA (903 mg, 7 mmol), and2-indoleacetic acid (307 mg, 1.75 mmol) in 5 ml DMF was stirred at 60°C. overnight. After extraction by EA, the target compound 5 (600 mg,84%) was afforded after purification by column with EA.

Step 5: To a solution of compound 5 (600 mg, 1.47 mmol) in EtOH/THF wasadded 2N NaOH (5 ml), then the resulting reaction mixture was stirred at60° C. overnight. After the solvent was evaporated off, the mixture wasextracted by EA, and then the combined organic layer was dried to affordthe desired compound 6 as a white solid (140 mg, 25%).

Step 6: A mixture of compound 6 (200 mg, 0.33 mmol), EDCI (102 mg, 0.66mmol), HOAT (88 mg, 0.66 mmol), DIPEA (170 mg, 1.32 mmol), and amine(102 mg, 0.33 mmol) in 5 ml DMF was stirred at 60° C. overnight. Afterextraction by EA, the target compound 7 (200 mg, 20%) was afforded as awhite solid.

Step 7: To a mixture of compound 7 (200 mg, 0.28 mmol) in 5 ml CH2Cl2was added 2 ml TFA, and the mixture was stirred at room temperature for2 h. After extraction by EA, the target Compound 034 (10 mg, 6%) waspurified by Prep-HPLC. ¹H NMR (500 MHz, DMSO) δ 10.86 (s, 1H), 9.67 (s,1H), 8.85 (s, 1H), 7.57-7.52 (m, 2H), 7.47.41 (m, 3H), 7.40-7.35 (m,3H), 7.33 (d, J=8.1 Hz, 1H), 7.26 (t, J=7.4 Hz, 1H), 7.12 (s, 1H), 7.09(d, J=8.8 Hz, 1H), 7.07-7.04 (m, 2H), 6.92 (t, J=7.0 Hz, 1H), 4.96 (s,1H), 4.49 (d, J=10.4 Hz, 1H), 4.12 (d, J=10.9 Hz, 1H), 3.71 (q, J=15.0Hz, 2H), 3.44 (q, J=7.0 Hz, 2H), 3.10 (t, J=12.3 Hz, 1H), 2.64 (t,J=12.2 Hz, 1H), 2.09 (s, 2H), 1.87 (dd, J=26.7, 12.8 Hz, 2H), 1.16-1.08(m, 1H), 1.05 (t, J=7.0 Hz, 3H). LCMS: m/z=622 (M+H)⁺.

Example 35: Synthesis of Compound 035

Step 1: A mixture of ethyl 2-chloropyrimidine-5-carboxylate (1.86 g, 10mmol), compound 1 (3.00, 15 mmol), and NEt3 (3.0 g, 30 mmol) in1,4-dioxane (20 ml) was stirred at 95° C. overnight. The mixture wasconcentrated, and EA (60 ml) and aqueous citric acid (60 ml) were added.The resulting reaction mixture was stirred for 30 min. The organic layerwas separated, dried and concentrated to afford compound 2 (3.4 g,yield: 970%) as alight yellow solid.

Step 2: Toa solution of compound 2 (600 mg, 1.7 mmol) in 1,4-dioxane (15ml) was added 4M HCl in 1,4-dioxane (10 ml) at room temperature, and theresulting reaction mixture was stirred overnight. The mixture wasfiltered to yield compound 3 (427 mg, 1000%) as a yellow solid.

Step 3: A mixture of compound 3 (427 mg, 1.7 mmol), compound2-indoleacetic acid (301 mg, 1.7 mmol), HOAT (462 mg, 3.4 mmol), EDCI(649 mg, 3.4 mmol) and NEt3 (687 mg, 6.8 mmol) in DMF (15 ml) wasstirred at 60° C. overnight. To the mixture was added EA (100 ml) andaqueous saturated NaCl (100 ml), and the resulting reaction mixture wasstirred for 30 min. The organic layer was separated, washed by aqueoussaturated NaCl (50 ml*2), dried and concentrated to yield a residue,which was purified by Prep-TLC to afford compound 4 (584 mg, 84%) as ayellow solid.

Step 4: To a solution of compound 4 (500 mg, 1.2 mmol) in EtOH (15 ml)and THE (15 ml) was added aqueous NaOH (2M, 15 mL), and the resultingreaction mixture was stirred at 60° C. for 2 h. The mixture wasconcentrated to afford a residue. To the mixture was added water (100mL) and aqueous citric acid to adjust to pH<7 at 0° C. followed byfiltration to yield compound 5 (342 mg, 75%) as a white solid.

Step 5: A mixture of compound 5 (150 mg, 0.39 mmol), compound Boc-amine(113 mg, 0.39 mmol), HOAT (103 mg, 0.76 mmol), EDCI (145 mg, 0.76 mmol)and NEt3 (154 mg, 1.52 mmol) in DMF (5 ml) was stirred at 60° C.overnight. To the mixture was added EA (100 ml) and aqueous saturatedNaCl (100 ml), and the resulting reaction mixture was stirred for 30min. The organic layer was separated, washed by aqueous saturated NaCl(50 ml*2), dried and concentrated to yield a residue, which was purifiedby Prep-TLC to afford compound 6 (150 mg, 59%) as a yellow solid.

Step 6: To a solution of compound 6 (150 mg, 0.23 mmol) in DCM (5 ml)was added TFA (3 ml) at room temperature, and the resulting reactionmixture was stirred for 2 h. The mixture was concentrated and purifiedby Prep-HPLC to afford Compound 035 (2 mg, 1.5%) as a white solid. ¹HNMR (400 MHz, DMSO) δ 10.92 (s, 1H), 9.57 (s, 1H), 8.86 (s, 2H), 7.84(d, J=7.8 Hz, 1H), 7.56 (dd, J=12.6, 7.9 Hz, 4H), 7.49 (s, 1H), 7.36(dt, J=17.7, 8.5 Hz, 5H), 7.27-7.19 (m, 3H), 7.07 (t, J=7.4 Hz, 1H),6.97 (t, J=7.3 Hz, 1H), 6.84 (d, J=8.3 Hz, 1H), 5.15 (s, 2H), 4.33 (d,J=14.0 Hz, 1H), 3.78 (d, J=2.4 Hz, 2H), 3.15 (t, J=12.5 Hz, 1H), 2.77(t, J=11.7 Hz, 1H), 1.84 (s, 2H), 1.32 (d, J=10.9 Hz, 2H). LCMS: m/z=548(M+H)⁺.

Example 36: Synthesis of Compound 036

Steps 1-3: Refer to steps 1-3 of Example 34 to obtain compound 4.

Step 4: A mixture of compound 4 (500 mg, 1.53 mmol), EDCI (573 mg, 3mmol), HOAT (405 mg, 3 mmol), DIPEA (390 mg, 3 mmol), and2-methyl-3-indoleacetic acid (289 mg, 1.53 mmol) in 10 ml DMF wasstirred at 60° C. overnight. After quenching with ice water, the targetcompound 5 (508 mg, 69%) was precipitated and was collected as a whitesolid.

Step 5: To a solution of compound 5 (508 mg, 1 mmol) in EtOH/THF (10 ml)was added 2N NaOH (5 ml), then the resulting reaction mixture wasstirred at 60° C. overnight. After the solvent was evaporated off, thepH of the mixture was adjusted to 4-5. The precipitate was collected toafford the desired compound 6 as a white solid (460 mg, 96%).

Step 6: A mixture of compound 6 (200 mg, 0.42 mmol), EDCI (160 mg, 0.84mmol), HOAT (113 mg, 0.84 mmol), DIPEA (108 mg, 0.84 mmol), and amine(120 mg, 0.42 mmol) in 5 ml DMF was stirred at 60° C. overnight. Afterextraction by EA, the target compound 7 (200 mg, crude) was afforded asan oil.

Step 7: To a mixture of compound 7 (200 mg, crude) in 5 ml CH2Cl2 wasadded 2 ml TFA, and the mixture was stirred at room temperature for 2 h.After extraction by EA, the target Compound 036 (94 mg, 46%) waspurified by Prep-HPLC. ¹H NMR (500 MHz, DMSO) δ 10.74 (s, 1H), 9.78 (s,1H), 8.84 (s, 2H), 7.61-7.54 (m, 3H), 7.48-7.36 (m, 6H), 7.34-7.25 (m,2H), 7.22 (d, J=8.0 Hz, 1H), 7.04-6.94 (m, 4H), 6.86 (t, J=7.4 Hz, 1H),4.93 (t, J=11.9 Hz, 1H), 4.48 (d, J=13.2 Hz, 1H), 3.98 (d, J=12.1 Hz,1H), 3.64 (dd, J=45.6, 15.4 Hz, 2H), 3.07 (t, J=12.4 Hz, 1H), 2.62 (t,J=12.2 Hz, 1H), 2.22 (s, 3H), 1.85 (d, J=11.3 Hz, 1H), 1.75 (d, J=10.8Hz, 1H), 1.08 (d, J=8.4 Hz, 1H), 0.94 (d, J=8.5 Hz, 1H). LCMS: m/z=636(M+H)⁺.

Example 37: Synthesis of Compound 037

Step 1: Refer to step 1 of Example 13 to obtain compound 5.

Step 2: A mixture of compound 5 (250 mg, 0.69 mmol) and NBS (123 mg, 069mmol in DCM (10 ml) was stirred at 0° C. for 30 min. To the mixture wasadded EA (20 ml) and aqueous saturated Na2SO3 (20 ml), and the resultingreaction mixture was stirred for 30 min. The organic layer wasseparated, washed by aqueous saturated NaCl (20 ml×2), dried andconcentrated, and purified by Prep-TLC to yield compound 6 (258 mg, 85%)as a yellow solid.

Step 3: A mixture of compound 6 (240 mg, 0.55 mmol), cyclopropylboronicacid (232 mg, 2.7 mmol), Pd(OAc)2 (11 mg, 0.05 mmol),tricyclohexylphosphine (14 mg, 0.05 mmol) and K3PO4 (360 mg, 1.7 mmol)in toluene (30 ml) and H2O (5 ml) was stirred at 95° C. under nitrogenatmosphere overnight. The mixture was cooled, and to the mixture wasadded EA (100 ml). Filtration, concentration, and purification by silicagel column yielded compound 7 (200 mg, 90%) as a light yellow solid.

Step 4: To a solution of compound 7 (200 mg, 0.5 mmol) in EtOH (15 mL)and THE (15 ml) was added aqueous NaOH (2M, 15 mL), and the resultingreaction mixture was stirred at 60° C. for 2 h. The mixture wasconcentrated, and then, to the mixture, was added water (20 ml) andaqueous citric acid to adjust to pH<7 at 0° C. followed by filtration toyield compound 8 (168 mg, 90%) as a white solid.

Step 5: A mixture of compound 8 (150 mg, 0.40 mmol), compound Boc-amine(114 mg, 0.40 mmol), HOAT (103 mg, 0.76 mmol), EDCI (145 mg, 0.76 mmol)and NEt3 (154 mg, 1.52 mmol) in DMF (5 ml) was stirred at 60° C.overnight. The mixture was extracted with EA (10 ml*2), dried andconcentrated, and purified by Prep-TLC to afford compound 9 (120 mg,50%) as a yellow solid.

Step 6: To a solution of compound 9 (120 mg, 0.20 mmol) in DCM (5 ml)was added TFA (3 ml) at room temperature, and the resulting reactionmixture was stirred for 2 h. The mixture was concentrated to affordcompound 10 (80 mg, 100%) and was used in the next step without furtherpurification.

Step 7: To a solution of the compound 10 (80 mg, 0.20 mmol) and Et3N(106 mg, 1.05 mmol) in THE (5 ml) was added morpholine-4-carbonylchloride (45 mg, 0.30 mmol) at 0° C., and the resulting reaction mixturewas stirred for 2 h. To the mixture was added EA (10 ml) and aqueoussaturated NaCl (10 ml), and the resulting reaction mixture was stirredfor 30 min. The organic layer was separated, concentrated, and purifiedby Prep-HPLC to afford Compound 037 (3 mg, 3%) as a yellow solid. ¹H NMR(400 MHz, MeOD) δ 7.78 (d, J=7.1 Hz, 2H), 7.58 (d, J=7.6 Hz, 2H), 7.48(s, 1H), 7.39 (t, J=7.9 Hz, 3H), 7.26 (t, J=7.4 Hz, 1H), 6.99 (d, J=8.3Hz, 1H), 6.93 (d, J=9.2 Hz, 1H), 3.71-3.66 (m, 4H), 3.50 (d, J=13.6 Hz,2H), 3.30-3.20 (m, 6H), 2.19 (d, J=13.9 Hz, 2H), 2.05 (s, 1H), 1.84-1.67(m, 3H), 1.53 (s, 3H), 1.33 (d, J=17.9 Hz, 1H), 1.06-0.99 (m, 2H), 0.67(d, J=4.4 Hz, 2H). LCMS: m/z=554 (M+H)⁺.

Example 38: Synthesis of Compound 038

Step 1: A mixture of the compound 1 (1.5 g, 10.2 mmol), (Boc)2O (2.2 g,10.2 mmol) and TEA (2.06 g, 20.4 mmol) in CH2Cl2 (50 ml) was stirred atroom temperature overnight. Concentration and purification by silica gelcolumn with EA:PE=1:5 afforded compound 2 (1.5 g, 60%) as a yellowsolid.

Step 2: The mixture of the compound 2 (1.4 g, 5.7 mmol), NaBH3CN (539mg, 8.6 mmol) and ammonium acetate (3.1 g, 40.0 mmol) in MeOH (20 ml)was stirred at 70° C. for 2 hours. Concentration and purification bysilica gel column with EA:PE=1:1 afforded compound 3 (1.2 g, 85%) as ayellow solid.

Step 3: S solution of the compound 3 (800 mg, 3.2 mmol), ethyl2-chloropyrimidine-5-carboxylate (603 mg, 3.2 mmol) and DIPEA (826 mg,6.4 mmol) in 1,4-dioxane (25 ml) was formed. The mixture was heated to95° C. and was stirred overnight. Concentration and purification bysilica gel column with EA:PE=1:10 afforded compound 4 (1.0 g, 79%) as alight yellow solid.

Step 4: A solution of the compound 4 (1.0 g, 2.5 mmol) and 2N NaOH (10ml, 20 mmol) in THF (10 ml) and EtOH (10 ml) was formed. The mixture washeated to 60° C. and was stirred for 6 hours. Concentration andadjustment of the pH of the water phase to 5-6 was followed byextraction with EA. The organic layers were washed with water and brinethen dried over sodium sulfate, filtered and concentrated to yieldtarget compound 5 (600 mg, 65%) as a white solid.

Step 5: A mixture of the compound 5 (100 mg, 0.27 mmol), tert-butyl3-aminobiphenyl-4-ylcarbamate (77 mg, 0.27 mmol), EDCI (53.0 mg, 0.41mmol), HOAT (55.0 mg, 0.41 mmol) and DIEA (70 mg, 0.54 mmol) in DMF (10ml) was formed. The mixture was heated to 65° C. and was stirredovernight. Then the mixture was dissolved in water and extracted withEA. The organic layers were washed with water and brine then dried oversodium sulfate, filtered and concentrated. Purification by silica gelcolumn with EA:PE=1:1 yielded compound 6 (130 mg, 76%) as a purplesolid.

Step 6: To a stirred solution of compound 6 (130 mg, 0.2 mmol) in CH2Cl2(10 ml) was added HCl/1,4-dioxane (2 ml, 8.0 mmol), and the resultingreaction mixture was stirred at room temperature overnight.Concentration and washing with PE afforded the target Compound 038 (55mg, 62%) as a white solid. ¹H NMR (400 MHz, DMSO) δ 9.58 (s, 1H), 8.92(s, 2H), 8.10 (d, J=8.8 Hz, 1H), 7.6-7.47 (m, 3H), 7.45-7.29 (m, 3H),7.24 (t, J=7.3 Hz, 1H), 6.98-6.81 (m, 3H), 6.54-6.40 (m, 2H), 5.85 (s,1H), 5.29 (s, 1H), 3.13-3.04 (m, 1H), 1.94 (d, J=5.1 Hz, 2H), 1.18 (t,J=7.2 Hz, 2H), 0.85 (s, 1H). LCMS: m/z=437 (M+H)⁺.

Example 39: Synthesis of Compound 039

Step 1: A mixture of compound 1 (200 mg, 0.62 mmol), EDCI (192 mg, 1.24mmol), HOAT (170 mg, 1.24 mmol), DIPEA (400 mg, 2.5 mmol), and amine(177 mg, 0.62 mmol) in 5 ml DMF was stirred at 60° C. overnight. Afterextraction by EA, the target compound 2 (220 mg, 59%) was afforded as acrude oil.

Step 2: To a mixture of compound 2 (220 mg, 0.4 mmol) in 5 ml CH2Cl2 wasadded 1 ml TFA. The mixture was stirred at room temperature for 2 h.After extraction by EA (10 ml) and water (10 ml*2), the target Compound039 (120 mg, 67%) was purified by Prep-HPLC. ¹H NMR (400 MHz, DMSO) δ9.65 (s, 1H), 8.89 (s, 2H), 8.58 (s, 1H), 8.32 (s, 1H), 8.07 (d, J=7.3Hz, 1H), 7.62-7.49 (m, 3H), 7.39 (dd, J=17.1, 9.2 Hz, 3H), 7.26 (t,J=7.4 Hz, 1H), 6.92 (s, 1H), 4.13 (s, 1H), 3.33 (d, J=12.8 Hz, 2H), 3.05(d, J=9.9 Hz, 2H), 2.05 (d, J=11.4 Hz, 2H), 1.77-1.63 (m, 2H). LCMS:m/z=389 (M+H)⁺.

Example 40: Synthesis of Compound 040

Step 1: A mixture of compound 1 (2.0 g, 17.5 mmol), ethyl2-chloropyrimidine-5-carboxylate (3.2 g, 17.5 mmol), and DIPEA (4.5 mg,35.0 mmol) in 5 ml CH2Cl2 was stirred at room temperature for 2 h. Afterextraction by EA (2*50 ml), the combined organic layer was dried toafford the target compound 2 (2.0 g, 43%) as a white solid.

Step 2: A solution of compound 2 (400 mg, 1.5 mmol) in 6M HCl wasstirred at 100° C. overnight, and the desired compound 3 was dried byfreeze dryer (300 mg, 85%).

Step 3: A mixture of compound 3 (150 mg, 0.63 mmol), EDCI (197 mg, 1.26mmol), HOAT (171 mg, 1.26 mmol), DIPEA (325 mg, 2.5 mmol), and amine(179 mg, 0.63 mmol) in 5 ml DMF was stirred at 60° C. overnight. Afterextraction by EA, the target compound (200 mg, 63%) was purified bycolumn with CH2Cl2:CH3OH (10:1).

Step 4: To a mixture of compound 4 (100 mg, 0.2 mmol) in 5 ml CH2Cl2 wasadded TFA (1 ml). The mixture was stirred at room temperature for 2 h.After extraction by EA (10 ml) and water (10 ml*2), the target Compound040 (20 mg, 25%) was purified by Prep-HPLC. ¹H NMR (400 MHz, DMSO) δ9.52 (s, 1H), 8.84 (s, 2H), 7.78 (d, J=7.5 Hz, 1H), 7.59-7.46 (m, 3H),7.39 (t, J=7.5 Hz, 2H), 7.32 (d, J=7.5 Hz, 1H), 7.24 (t, J=7.0 Hz, 1H),6.85 (d, J=8.3 Hz, 1H), 5.14 (s, 2H), 3.77 (s, 1H), 2.75 (d, J=11.5 Hz,2H), 2.16 (s, 3H), 1.95 (t, J=10.9 Hz, 2H), 1.83 (d, J=10.4 Hz, 2H),1.55 (d, J=9.5 Hz, 2H). LCMS: m/z=403 (M+H)⁺.

Example 41: Synthesis of Compound 041

Steps 1-5: Refer to steps 1-5 of Example 7 to obtain compound 6.

Step 6: A mixture of compound 6 (200 mg, 0.62 mmol), EDCI (192 mg, 1.24mmol), HOAT (170 mg, 1.24 mmol), DIPEA (400 mg, 2.5 mmol), and amine(177 mg, 0.62 mmol) in 5 ml DMF was stirred at 60° C. overnight. Afterextracted by EA, the target compound 7 (187 mg, 50%) was afforded as acrude oil.

Step 7: To a mixture of compound 7 (186 mg, 0.31 mmol) in 5 ml CH2Cl2was added 1 ml TFA. The mixture was stirred at room temperature for 2 h.After extraction by EA (10 ml) and water (10 ml*2), the target Compound041 (90 mg, 70%) was purified by Prep-HPLC. ¹H NMR (400 MHz, DMSO) δ9.57 (s, 1H), 8.88 (s, 2H), 7.63 (s, 1H), 7.54 (d, J=7.4 Hz, 2H), 7.49(s, 1H), 7.39 (t, J=7.7 Hz, 2H), 7.35-7.29 (m, 1H), 7.24 (t, J=7.3 Hz,1H), 6.85 (d, J=8.4 Hz, 1H), 5.16 (s, 2H), 3.04 (d, J=13.0 Hz, 2H), 2.96(t, J=10.8 Hz, 2H), 2.43 (s, 2H), 1.68 (t, J=10.7 Hz, 2H), 1.44 (s, 3H).LCMS: m/z=402 (M+H)⁺.

Example 42: Synthesis of Compound 042

Step 1: A mixture of compound 1 (2.0 g, 13.6 mmol), iodomethane (5.8 g,40.8 mmol), and K2CO3 (5.5 g, 40.8 mmol) in DMF (30 ml) was stirred at80° C. overnight. To the mixture was added EA (100 ml) and aqueoussaturated NaCl (100 ml), and the resulting reaction mixture was stirredfor 30 min. The organic layer was separated, washed by aqueous saturatedNaCl (50 ml*2), dried and concentrated, purified by silica gel column toafford compound 2 (1.5 g, 68%) as a yellow solid.

Step 2: A mixture of compound 2 (1.5 g, 9.3 mmol), NaBH3CN (1.8 g, 27.9mmol), and AcONH4 (3.6 g, 46.5 mmol) in MeOH (30 ml) was stirred at 70°C. for 5 h. To the mixture was added EA (100 ml) and aqueous saturatedNaCl (100 ml), and the resulting reaction mixture was stirred for 30min. The organic layer was separated, washed by aqueous saturated NaCl(50 ml*2), dried and concentrated to afford compound 3 (1.4 g, 80%) as awhite solid.

Step 3: A mixture of compound 3 (1.6 g, 10 mmol), ethyl2-chloropyrimidine-5-carboxylate (1.7 g, 9 mmol), and DIPEA (3.9 g, 30mmol) in DCM (30 ml) was stirred at room temperature overnight. Themixture was purified by silica gel column to afford compound 4 (800 mg,29%) as a yellow solid.

Step 4: To a solution of compound 4 (800 mg, 2.6 mmol) in EtOH (20 ml)and THE (20 ml) was added aqueous NaOH (2M, 20 ml). The mixture wasstirred at 60° C. for 2 h. The mixture was concentrated to yield aresidue, and, to the mixture, was added water (100 ml) and aqueouscitric acid to adjust to pH<7 at 0° C. followed by filtration to affordcompound 5 (600 mg, 80%) as a white solid.

Step 5: A mixture of compound 5 (150 mg, 0.53 mmol), compound Boc-amine(152 mg, 0.53 mmol), HOAT (103 mg, 0.76 mmol), EDCI (145 mg, 0.76 mmol)and NEt3 (154 mg, 1.52 mmol) in DMF (5 mL) was stirred at 60° C.overnight. The mixture was extracted by EA (10 ml) and water (10 ml*2).The organic layer was separated, washed by aqueous saturated NaCl (20ml*2), dried and concentrated, and purified by Prep-TLC to affordcompound 6 (45 mg, 15%) as a yellow solid.

Step 6: To a solution of compound 6 (45 mg, 0.08 mmol) in 1,4-dioxane (5ml) was added HCl in 1,4-dioxane (0.5 ml) at room temperature, and theresulting reaction mixture was stirred overnight. The mixture wasfiltered to afford Compound 042 (15 mg, 41%). ¹H NMR (500 MHz, DMSO) δ10.43 (s, 1H), 9.00 (d, J=29.0 Hz, 2H), 8.30 (d, J=8.6 Hz, 1H), 7.80 (d,J=1.9 Hz, 1H), 7.67 (d, J=7.4 Hz, 2H), 7.61 (dd, J=8.3, 1.9 Hz, 1H),7.49 (t, J=7.7 Hz, 2H), 7.45-7.37 (m, 2H), 7.12 (t, J=7.8 Hz, 1H), 7.04(d, J=7.3 Hz, 1H), 6.72 (d, J=8.2 Hz, 1H), 6.63 (t, J=7.3 Hz, 1H), 5.32(d, J=6.8 Hz, 1H), 3.39 (s, 2H), 2.90 (s, 3H), 2.07 (dd, J=22.1, 4.2 Hz,2H). LCMS: m/z=451 (M+H)⁺.

Example 43: HDAC Enzyme Assays

Compounds for testing were diluted in DMSO to 50 fold the finalconcentration and a ten point three fold dilution series was made. Thecompounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl,0.001% Tween-20, 0.05% BSA, 20 μM tris(2-carboxyethyl)phosphine) to 6fold their final concentration. The HDAC enzymes (purchased from BPSBiosciences) were diluted to 1.5 fold their final concentration in assaybuffer and pre-incubated with the compounds for 24 hours prior toaddition of the substrate.

The substrate tripeptide substrate 3 (synthesized in house) for eachenzyme was equal to the Km as determined by a substrate titration curve.The enzyme and substrate concentrations used are given in Table 2. Thesubstrates were diluted in assay buffer at 6× their final concentrationwith 0.3 μM sequencing grade trypsin (Sigma). The substrate/trypsin mixwas added to the enzyme/compound mix, the plate was shaken for 60seconds and placed into a Spectramax M5 microtiter plate reader. Thedevelopment of fluorescence was monitored for 30 min and the linear rateof the reaction was calculated. The IC₅₀ was determined using Graph PadPrism by a four parameter curve fit. The IC₅₀ values obtained for thecompounds of this invention are found in Table 1. Examples of the curvesare found in FIGS. 1 and 4 .

TABLE 2 Enzyme Substrate concentration concentration HDAC1  3.5 ng/μl3.8 μM HDAC2  0.2 ng/μl 2.3 μM HDAC3 0.08 ng/μl 3.9 μM

Example 44: Pharmacokinetics

Male SD rats were fasted overnight. Compounds of the invention weredissolved in dimethyl acetamide at 10 times the final concentration,then Solutol HS 15 (BASF) was added to a final concentration of 10%.Finally 80% saline was added and vortexed to achieve a clear solution.For the IV dosing three animals were injected via the foot dorsal veinwith 1 mg/kg compound. For the PO dosing 5 mg/kg of compound wasdelivered by oral gavage. Blood was collected via the tail vein intoK2EDTA tubes at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4hours, 8 hours and 24 hours after dosing. The blood was centrifuged at2000 g for 5 minutes at 4° C. to obtain plasma. The plasma was extractedwith acetonitrile and the level of compound was analyzed by LC/MS/MS.The level of compound in plasma was calculated from a standard curve inrat plasma. The IV clearance and area under the curve were calculatedusing WinNonLin software. The dose adjusted area under the curve for theIV and oral dosing were used to calculate the oral bioavailability.

A summary of results is presented in Table 3 and Table 4, as well asFIG. 2 .

Cynomolgus monkey pharmacokinetics was determined for Compound 005.Compound 005 was dissolved in 0.5% hydroxypropyl methyl cellulose andgiven to fed cynomolgus monkeys at 20 mg/kg body weight. Plasma sampleswere collected over time and analyzed as described above. Plasma levelsat each time point are shown in FIG. 5 .

TABLE 3 Cell based Cassette Rat PK (5 mg/kg PO) assay IV PO PO POCompound potency Clr. C_(max) T1/2 AUC F% Compound 003 3 μM 0.12 2316 1759876 118 Compound 001 3 μM 0.53 988 3.8 5959 63 Compound 002 3 μM 1.21011 5.9 6091 142 Compound 005 1 μM 0.38 569 >24 9162 79 Compound 004 ND0.57 1497 7.6 15941 209 Compound 006 ND 0.15 1517 14.5 31640 76Table 3: Compounds were tested in the fetal globin induction assay andthe lowest concentration to achieve a 2 fold increase in fetal globingene expression over baseline is presented in the “Cell based assaypotency” column. Pharmacokinetic properties were assessed in a ratcassette dosing experiment. The IV clearance (IV Clr.) is in units ofL/hr/kg. The oral maximum plasma concentration (PO Cmax) is in units ofng/ml. The oral plasma half life (PO T1/2) is in units of hours. Theoral area under the curve (PO AUC) is in units of hours*ng/ml. Thefraction absorbed by the oral route (F %) is a percentage of the oralarea under the curve to the IV area under the curve, dose adjusted.

TABLE 4 In vitro AD ME (Absorption, Distribution, Metabolism, andExcretion) In vitro tox Plasma Protein Microsome Compound hERG Cyp AmesMN Solubility stability binding stability Comp. 003 >30 μM >10 μM NegNeg 54 μM ND ND 4.7 min Comp. 001 >30 μM 2C9 ND ND 16.9 μM ND ND 11 minComp. 002 >30 μM >10 μM ND ND 42.7 μM ND ND ND Comp. 005 13.8 μM >10 μMNeg Neg 13.8 μM ND ND ND Comp. 004 12 μM >10 μM ND ND 12 μM ND ND NDComp. 006 >30 μM >10 μM ND ND ND ND ND ND

Example 45: Fetal Globin Induction

CD34+ cells isolated from human bone marrow were cultured in vitro usinga method described by Bradner J E (Proc Natl Acad Sci USA. 2010 Jul. 13;107(28):12617-22), which consists of a 7 day expansion phase in mediathat supports differentiation of cells towards the erythroid lineagefollowed by a differentiation phase for 3 days where erythroid celldevelopment continues. At the end of the differentiation period thesecells are primarily late erythroblasts. mRNA levels were determined byquantitative real time PCR using primer/probe sets designed to adultmajor β-globin (β), adult minor β-globin (δ), fetal p-like globin (HbG,γ), and embryonic β-like globin (ε). Protein levels were determined byflow cytometry using fluorescent antibodies against fetal hemoglobin(HbF) or adult hemoglobin (HbA).

In the experiment shown in FIG. 3 , cells were differentiated in thepresence of vehicle (DMSO) 0.3, 1 and 3 μM of Compound A, and of 0.3, 1,and 3 μM of Compound 003. In the experiment shown in FIG. 6 , cells weredifferentiated in the presence of vehicle (DMSO) 0.3, 1 and 3 μM ofCompound A, and of 0.3, 1, and 3 μM of Compound 005. Globin mRNA levelswere determined at day 3 of differentiation.

Compound A is an HDAC1/2 inhibitor (IC₅₀ is about 4, 15, and 114 nM forHDAC1, HDAC2, and HDAC3, respectively) with the structure shown below.The characterization and synthesis of Compound A is found in U.S.Publication No. 2014-0128391.

Example 46: Additional Studies

Additional experiments were performed that show Compound 003 lacks hERG,CYP inhibition, and genotoxicity.

For hERG assays, a CHO cell line stably transfected with hERG cDNA andexpressing hERG channels were seeded into a QPatch plate (Sophion) at adensity of 3-8×10⁶ cells/ml. The cells were voltage clamped at a holdingpotential of −80 mV. The hERG current was activated by depolarizing at+20 mV for 5 sec, after which the current was taken back to −50 mV for 5sec to remove the inactivation and observe the deactivating tailcurrent. The maximum amount of tail current size was used to determinehERG current amplitude. Six doses (30, 10, 3, 1, 0.3 and 0.1 μM) ofCompound 003 were chosen to obtain fitting curves and IC50. Data wereanalyzed using Assay Software provided by Sophion and Graphpad Prism.Compound 003 did not inhibit the activity of the hERG channel at thehighest concentration of 30 μM.

For Cyp inhibition, human live microsomes from BD Gentest were incubatedwith Compound 003 (10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01 μM) andsubstrate (CYP1A2: Phenacetin at 30 μM; CYP2C9: Diclofenac at 10 μM;CYP2C19: S-Mephenytoin at 35 μM; CYP3A4: Midazolam at 5 μM andTestosterone at 80 μM; CYP2D6: Bufuralol at 10 μM) for the followingincubation times: CYP1A2, 2C9, 2D6: 10 minutes, 37° C.; CYP2C19: 45minutes, 37° C.; CYP3A4: 5 minutes, 37° C. Substrate conversion wasmeasured by liquid chromatography/mass spectrometry/mass spectrometry(LC/MS/MS). Inhibition was calculated by curve fitting in Graph PadPrism. Compound 003 did not inhibit activity of any Cyp up to 10 M.

For genotoxicity, bacterial tester strains TA98, TA100, TA1535 and TA97aas described by Ames et al. (1975) and the E. coli tester strain WP2uvrA as described by Green and Muriel (1976) were incubated withCompound 003 at 250, 75, 25, 7.5, 2.5, 0.75, 0.25, and 0.075 g/well in24 well plates either with or without liver homogenate (S9) purchasedcommercially (MoTox; Boone, N.C.) prepared from male Sprague Dawley ratsthat have been injected intraperitonealy with Aroclor 1254 (200 mg/mL incorn oil), at a dose of 500 mg/kg, 5 days before sacrifice. Mutagenicityis evaluated by counting the number of colonies that form onnon-permissive media. Compound 003 did not increase the number ofrevertant colonies of any strain either with or without S9 activation.

Genotoxicity was further evaluated by micronucleus formation assay inhuman peripheral blood lymphocytes (HPBL). HPBL were obtained fromhealthy donors and exposed to Compound 003 at 3500, 2450, 1715, 1200,840, 588, 412, 288, 202, 141, 98.8, 69.2, 48.4, and 33.9 μg/ml for 4hours with or without S9 activation. The cells were washed with PBS andincubated in complete medium with 6 g/ml cytocholasin B for 24 hours.The cells were then lysed, fixed, and mounted on microscope slides. Thenumber of mononucleated, binucleated, and micronucleated cells wascounted under blinded conditions. Compound 003 did not increase thenumber of micronucleated cells at any concentration.

Example 47: Treatment of Erythroid Progenitors with Various HDAC1/2Inhibitors Leads to Induction of Gata2 mRNA

Human bone marrow derived CD34+ cells were expanded for 7 days asdescribed by Sankaran et al., Science, vol. 322(5909), pp. 1839-42(2008). Cells were then differentiated, in the presence of the indicatedconcentration of Compound 005, Compound A (another known HDAC1/2inhibitor), or vehicle control (DMSO), for 3 days in media supportingerythropoiesis (Hu et al., “Isolation and functional characterization ofhuman erythroblasts at distinct stages: implications for understandingof normal and disordered erythropoiesis in vivo”, Blood, vol. 121(16),pp. 3246-53 (2005)). Gata2 mRNA was determined using quantitative realtime PCR and expressed relative to the level beta-actin mRNA control.Compound 005 or Compound A treatment of primary erythroid progenitorsresults in an equivalent dose and time-dependent induction of % HbG(FIG. 6 ) and Gata2 mRNA (FIG. 7 ).

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties. Unless otherwise defined, alltechnical and scientific terms used herein are accorded the meaningcommonly known to one with ordinary skill in the art.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method for treating a disease mediated by HDAC1 and/orHDAC2 in a subject comprising administering to the subject a compound ofFormula I:

or a pharmaceutically acceptable salt thereof, wherein, X¹ is CR⁷ or N;X² is CH or N; wherein X¹ and X² are each N, or X¹ and X² are each CH; Yis selected from the group consisting of:

Z is selected from the group consisting of H, C₁-C₆-alkyl, C₆-aryl,C(O)NR⁴R⁵, C(O)OR⁶, C(O)C₁-C₆-alkyl, C(O)C₀-C₆-alkyl-C₆-aryl,C(O)—C₃-C₆-cycloalkyl, C(O)—C₂-C₆-heterocyclyl, andC(O)C₀₋₆-alkyl-heteroaryl, wherein the aryl, heteroaryl, cycloalkyl, andheterocyclyl groups are optionally substituted by 1 or 2 of C₁-C₆-alkyl,halo, C₁-C₆-haloalkyl, hydroxy, or C₁-C₆-alkoxy; R^(a) and R^(b) are H,or R^(a) and R^(b) together form a fused C₆-aryl; R¹ is selected fromthe group consisting of H and C₁-C₆-alkyl; R² is selected from the groupconsisting of H, C₁-C₆-alkyl, and C₆-aryl; R³ is selected from the groupconsisting of H, C₁-C₆-alkyl, and C₆-aryl; or R² and R³ together form aC₂-C₆-heterocyclyl; R⁴ is selected from the group consisting of H,C₁-C₆-alkyl, C₁-C₆-alkyl-OH, and C₁-C₆—NH₂; R⁵ is C₁-C₆-alkyl; or R⁴ andR⁵ together form a C₂-C₆-heterocyclyl, wherein heterocyclyl isoptionally substituted by 1 or 2 of C₁-C₆-alkyl, halo, C₁-C₆-haloalkyl,hydroxy, or C₁-C₆-alkoxy; R⁶ is selected from the group consisting ofC₁-C₆-alkyl and C₀-C₆-alkyl-C₆-aryl, wherein aryl is optionallysubstituted by 1 or 2 of C₁-C₆-alkyl, halo, or hydroxy; and R⁷ is H;wherein the disease mediated by HDAC1 and/or HDAC2 is selected from thegroup consisting of sickle-cell disease, beta-thalassemia, lung cancer,colon cancer, neuroblastoma, leukemia, and lymphoma.
 2. The method ofclaim 1, having the structure of Formula II:

or a pharmaceutically acceptable salt thereof.
 3. The method of claim 1,wherein Z is selected from the group consisting of C(O)NR⁴R⁵, C(O)OR⁶,C(O)—C₃-C₆-cycloalkyl, C(O)—C₂-C₆-heterocyclyl, andC(O)C₀₋₆-alkyl-heteroaryl, wherein heteroaryl, cycloalkyl, orheterocyclyl are optionally substituted by 1 or 2 of C₁-C₆-alkyl, halo,or hydroxy; and R⁶ is C₆-aryl.
 4. The method of claim 1, wherein Z isselected from the group consisting of H, C₁-C₆-alkyl, and C₆-aryl. 5.The method of claim 1, wherein R¹ is H.
 6. The method of claim 1,wherein R² is H.
 7. The method of claim 1, wherein R³ is H, methyl,ethyl, isopropyl, or phenyl.
 8. The method of claim 1, wherein thecompound of Formula I is a compound of Formula III:

or a pharmaceutically acceptable salt thereof.
 9. The method of claim 8,wherein R² is H.
 10. The method of claim 8, wherein R³ is H, methyl, orisopropyl.
 11. The method of claim 8, wherein R⁴ is H and R⁵ isC₁-C₆-alkyl.
 12. The method of claim 8, wherein R⁴ and R⁵ together forma heterocyclyl selected from the group consisting of morpholinyl,piperidinyl, piperazinyl, and pyrrolidinyl, wherein the morpholinyl,piperidinyl, piperazinyl, and pyrrolidinyl are optionally substituted by1 or 2 of C₁-C₆-alkyl, halo, or hydroxy.
 13. The method of claim 8,wherein X¹ and X² are N; R¹ is H; R² is H; R³ is H or C₁-C₄-alkyl; andR⁴ and R⁵ together form a heterocyclyl selected from the groupconsisting of morpholinyl, piperidinyl, piperazinyl, and pyrrolidinyl,wherein the morpholinyl, piperidinyl, piperazinyl, and pyrrolidinyl areoptionally substituted by 1 or 2 of C₁-C₆-alkyl, halo, or hydroxy. 14.The method of claim 8 wherein the compound of Formula III is selectedfrom:

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or pharmaceutically acceptable salts thereof.
 15. The method of claim 8wherein the compound of Formula III is selected from:

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or pharmaceutically acceptable salts thereof.
 16. The method of claim 1,wherein the hemoglobinopathy is sickle-cell disease or beta-thalassemia.17. The method of claim 1, wherein the disease is lung cancer, coloncancer, neuroblastoma, leukemia, or lymphoma.
 18. The method of claim 1,wherein the disease is neuroblastoma.
 19. The method of claim 1, whereinthe subject is a human.